Carbon fixation systems in plants and algae

ABSTRACT

Provided are heterologous nucleic acid constructs, vectors and methods for elevating cyclic electron transfer activity, improving carbon concentration, and enhancing carbon fixation in C3 and C4 plants, and algae, and producing biomass or other products from C3 or C4 plants, and algae, selected from among, for example, starches, oils, fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids, as well as transgenic plants produced thereby. These methods and transgenic plants and algae encompass the expression, or overexpression, of various combinations of genes that improve carbon concentrating systems in plants and algae, such as bicarbonate transport proteins, carbonic anhydrase, light driven proton pump, cyclic electron flow regulators, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/358,331, titled “Improved Carbon Fixation Systems in Plants and Algae”, filed on Mar. 3, 2019, which claims priority to U.S. patent application Ser. No. 15/411,854, entitled “Improved Carbon Fixation Systems in Plants and Algae”, filed on Jan. 20, 2017, and issued on Mar. 3, 2019 as U.S. Pat. No. 10,233,458, which is a continuation of International Patent Application No. PCT/US2015/041617, entitled “Improved Carbon Fixation Systems in Plants and Algae”, filed on Jul. 22, 2015, which claims priority to and the benefit of the filing of U.S. Provisional Patent Application No. 62/027,354, entitled “Carbon Fixation Systems in Plants and Algae”, filed on Jul. 22, 2014, and the specification and claims thereof are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants Nos. DOE-CECO Prime No: DE-AR0000202, Sub No: 21018-N; DOE-CABS Prime No: DE-SC0001295, Sub No: 21017-NM NSF EF-1219603, NSF No:1219603. The U.S. government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 5, 2017, is named 040517_NMC0001-101-US_Sequence_Listing_ST25.txt and is 286 KB in size.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

COPYRIGHTED MATERIAL

Not Applicable.

BACKGROUND

A major factor limiting photosynthetic efficiency is the competitive inhibition of CO₂ fixation by oxygen, due to lack of specificity of the enzyme RuBisCO. Incorporation of oxygen by RuBisCO is the first-dedicated step in photorespiration, a pathway that respires CO₂, compounding photosynthetic inefficiency. Overall, photorespiration reduces photosynthetic productivity by as much as 50% [1]. To date, attempts to engineer reduced oxygenase activity in RuBisCO have been largely unsuccessful.

Significantly, the cyanobacteria, eukaryotic microalgae, and C4 plants have evolved mechanisms to reduce photorespiration by concentrating CO₂ near RuBisCO, competitively inhibiting oxygenase activity and leading to substantial increases in yield and water use efficiency per unit carbon fixed. However, carbon concentrating systems (CCMs) are not operational in the vast majority of plant species (i.e., C3 plants).

Attempts to reconstitute functional CCMs in C3 plants have been previously attempted by us and others, mainly focusing on engineering pathways that are directly involved in facilitating CO₂ transport into leaf chloroplasts. Note, for example, PCT International Publication WO 2012/125737; Sage and Sage (2009) Plant and Cell Physiol. 50(4):756-772; Zhu et al. (2010) J Interg. Plant Biol. 52(8):762-770; Furbank et al. (2009) Funct. Plant Biol. 36(11):845-856; Weber and von Caemmerer (2010) Curr. Opin. Plant Biol.; Price (2013) J. Exp. Bot. 64(3):753-68; and U.S. Patent Application Publication No. 2013/0007916 A1.

However, ATP and NADPH production through light harvesting and electron transfer steps must be coordinated with carbon assimilation and additional energy requiring steps including CCM systems to prevent photoinhibition and to improve growth. Additionally, assimilatory flux and storage rates can limit carbon fixation due to feedback inhibition when sink demand is not matched to source capacity [2].

Thus, there is a critical need to improve plant productivity through integrated systems engineering approaches that balance source/sink interactions with energy and reductant production to develop energy-requiring, artificial CCMs that can effectively mimic those found in nature.

BRIEF SUMMARY OF THE INVENTION

Accordingly, in response to this need, the present disclosure provides methods for elevating cyclic electron transfer activity, improving carbon concentration, and enhancing carbon fixation in C3 and C4 plants, and algae, and producing biomass or other products from C3 or C4 plants, and algae, selected from among, for example, starches, oils, fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids, as well as transgenic plants produced thereby. These methods and transgenic plants and algae encompass the expression, or overexpression, of various combinations of genes that improve carbon concentrating systems in plants and algae, such as bicarbonate transport proteins, carbonic anhydrase, light driven proton pump, cyclic electron flow regulators, etc. Thus, among its various embodiments, the present disclosure provides the following:

A first embodiment of the present invention provides for a transgenic plant or alga, comprising within its genome, and expressing or overexpressing, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane and a cyclic electron transfer modulator protein. The cyclic electron transfer modulator protein may be selected from a PGRL1 protein (for example SEQ ID NO:3), a PGR5 protein (for example SEQ ID NO:1), a leaf FNR1 protein (for example SEQ ID NO:96), a leaf FNR2 protein (for example SEQ ID NO:97), a Fd1 protein (for example SEQ ID NO:95), or any combination thereof and for example the ATP dependent bicarbonate anion transporter localized to the plasma membrane may be a HLA3 protein (for example SEQ ID NO:77). The transgenic plant or alga described may further comprise within its genome, and expressing or overexpressing the heterologous nucleotide sequence encoding a bicarbonate anion transporter protein localized to the chloroplast envelope. The transgenic plant or alga described herein may further comprise within its genome, and expressing or overexpressing the heterologous nucleotide sequence a carbonic anhydrase protein. In a preferred embodiment, the cyclic electron transfer modulator protein is a PGR5 protein, in another preferred embodiment the cyclic electron transfer modulator protein is Fd1 protein, in yet another preferred embodiment, in still another preferred embodiment the cyclic electron transfer modulator protein is leaf FNR1, in a further preferred embodiment the cyclic electron transfer modulator protein is PGRL1. In a preferred embodiment the heterologous nucleotide sequences of the transgenic plant or alga encode i) a PGR5 protein, and a HLA3 protein; or ii) a PGR5 protein, a HLA3 protein and a PGRL1 protein ora PGR5 protein, a HLA3 protein, and a LCIA protein or a PGR5 protein, a HLA3 protein, a PGRL1 protein, a LCIA protein, and a BCA or HCA2 protein. In another preferred embodiment the heterologous nucleotide sequences the transgenic plant or alga of wherein encode a PGR5 protein, a HLA3 protein, a LCIA protein and a BCA or optionally a HCA2 protein. The transgenic plant or alga as described wherein the PGR5 protein has an amino acid sequence at least 80% identical to SEQ ID NO:1; the HLA3 protein has an amino acid sequence at least 80% identical to SEQ ID NO:77; the PGRL1 protein has an amino acid sequence at least 80% identical to SEQ ID NO:3; the LCIA protein has an amino acid sequence at least 80% identical to SEQ ID NO:18; and/or the BCA protein has an amino acid sequence at least 80% identical to SEQ ID NO:21. Alternatively, the sequence identity/sequence similarity is about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to those specifically disclosed which includes for example proteins without a transit peptide sequence and the functional protein.

A second embodiment provides for a transgenic plant or alga, comprising within its genome, and expressing or overexpressing, a combination of heterologous nucleotide sequences encoding:

LCIA protein and BCA protein or HCA protein is provided. In a preferred embodiment the heterologous nucleotide sequences encode transgenic plant or alga wherein the LCIA protein has an amino acid sequence at least 80% identical to SEQ ID NO:18; and/or the BCA protein has an amino acid sequence at least 80% identical to SEQ ID NO:21 and the HCA protein has an amino acid sequence at least 80% identical to SEQ ID NO:19. Alternatively, the sequence identity/sequence similarity is about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to those specifically disclosed which include for example proteins without a transit peptide sequence and the functional protein.

A third embodiment provides for a transgenic plant or alga, comprising within its genome, and expressing or overexpressing, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane (for example SEQ ID NO:77), a bicarbonate anion transporter localized to the chloroplast envelope (for example SEQ ID NO:18), a carbonic anhydrase, a proteorhodopsin protein targeted to thylakoid membranes (for example SEQ ID NO:98), and a R carotene monooxygenase protein (for example SEQ ID NO:100). In another preferred embodiment the proteorhodopsin comprises a chloroplast transit peptide selected from among a psbX stop-transfer trans-membrane domain fused to its C-terminus, a DNAJ transit peptide, a CAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide. In another preferred embodiment the β-carotene monooxygenase is expressed under the control of a promoter selected from among an ethanol inducible gene promoter and a green tissue/leaf-specific promoter selected from among CAB and rbcS. The proteorhodopsin may comprise an amino acid substitution selected from among L219E/T206S, M79T, and M79Y, and combinations thereof.

The carbonic anhydrase of the first, second, or third embodiment may be a BCA or optionally a HCA2 protein. The bicarbonate anion transporter localized to the chloroplast envelope of the first, second and third embodiment may be a LCIA protein. The ATP dependent bicarbonate anion transporter localized to the plasma membrane of the first and third embodiments may be HLA3.

A fourth embodiment provides for a method of making a transgenic plant or alga of a first embodiment wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane and a cyclic electron transfer modulator protein. The cyclic electron transfer modulator protein may be selected from a PGRL1 protein, a PGR5 protein, a FNR1 protein, a FNR2 protein (leaf-form isotopes), a Fd1 protein, or any combination thereof and wherein the ATP dependent bicarbonate anion transporter localized to the plasma membrane is a HLA3 protein. The heterologous nucleotide sequences of the fourth embodiment further encoding a bicarbonate anion transporter protein localized to the chloroplast envelope for example the bicarbonate anion transporter protein is LCIA. Additionally, the heterologous nucleotide sequences encode a carbonic anhydrase protein for example a BCA protein or optionally a HCA2 protein. In a preferred embodiment the cyclic electron transfer modulator protein is a PGR5 protein and optionally a PGRL1 protein and or combination thereof.

A fifth embodiment provides a method of making a transgenic plant or alga as described in a second embodiment, wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding a LCIA protein and a BCA protein or optionally a HCA protein.

A sixth embodiment provides a method of making a transgenic plant or alga of a third embodiment wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane, a bicarbonate anion transporter, a carbonic anhydrase, a proteorhodopsin protein targeted to thylakoid membranes, and a R carotene monooxygenase protein. In a preferred embodiment the proteorhodopsin comprises a chloroplast transit peptide selected from among a psbX stop-transfer trans-membrane domain fused to its C-terminus, a DNAJ transit peptide, a CAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide. In another preferred embodiment the β-carotene monooxygenase is expressed under the control of a promoter selected from among an ethanol inducible gene promoter and a green tissue/leaf-specific promoter selected from among CAB and rbcS. In a preferred embodiment the proteorhodopsin comprises an amino acid substitution selected from among L219E/T206S, M79T, and M79Y, and combinations thereof. In another preferred embodiment the ATP dependent bicarbonate anion transporter localized to the plasma membrane is HLA3.

The transgenic plant of an embodiment disclosed herein may be a C3 plant or a C4 plant such as a transgenic oilseed plant or a transgenic food crop plant which may include the genera Brassica (e.g., rapeseed/canola (Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea), Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis), coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard other than Arabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm; palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis; Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphea lanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina; Vernonia galamensis; Momordica charantia; and Crambe abyssinica, wheat, rice, maize (corn), barley, oats, sorghum, rye, and millet; peanuts, chickpeas, lentils, kidney beans, soybeans, lima beans; potatoes, sweet potatoes, and cassavas; soybeans, corn, canola, peanuts, palm, coconuts, safflower, cottonseed, sunflower, flax, olive, and safflower; sugar cane and sugar beets; bananas, oranges, apples, pears, breadfruit, pineapples, and cherries; tomatoes, lettuce, carrots, melons, strawberry, asparagus, broccoli, peas, kale, cashews, peanuts, walnuts, pistachio nuts, almonds; forage and turf grasses; alfalfa, clover; coffee, cocoa, kola nut, poppy; vanilla, sage, thyme, anise, saffron, menthol, peppermint, spearmint and coriander and preferably wheat, rice and canola. The transgenic alga of an embodiment disclosed herein may be selected from among a Chlorella species, a Nannochloropsis species, and a Chlamydomonas species. The heterologous nucleotide sequences are described in an embodiment may be codon-optimized for expression in said transgenic plant or alga. One aspect of the present invention provides for a transgenic plant or alga as described in an embodiment which exhibits enhanced CO₂ fixation compared to an otherwise identical control plant grown under the same conditions for example wherein CO₂ fixation is enhanced in the range of from about 10% to about 50% compared to that of an otherwise identical control plant grown under the same conditions.

A fourth embodiment provides for a part of said transgenic plant or alga of any embodiment described herein. For example, the part of said transgenic plant may be selected from among a protoplast, a cell, a tissue, an organ, a cutting, an explant, a reproductive tissue, a vegetative tissue, biomass, an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, a callus, propagation materials, germplasm, cuttings, divisions, and propagations.

A fifth embodiment provides for a progeny or derivative of said transgenic plant or alga of any embodiment described herein. For example, the progeny or derivatives may be selected from among clones, hybrids, samples, seeds, and harvested material thereof and may be produced sexually or asexually.

Another embodiment of the present invention provides a method of elevating CET activity in a C3 plant, C4 plant, or alga wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane and cyclic electron transfer modulator protein.

Yet another embodiment provides a method of enhancing carbon fixation in a C3 plant, C4 plant, or alga wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane and a cyclic electron transfer modulator protein.

Yet another method provides for a method of producing biomass or other products from a C3 plant, C4 plant, or an alga, wherein said products are selected from among starches, oils, fatty acids, triacylglycerols, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane and a cyclic electron transfer modulator protein. This method further comprises growing said plant or alga and harvesting said biomass or recovering said product from said plant or alga. Another aspect of the present invention provides for biomass or other product produced from a plant or alga selected from among starches, oils, fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids, made by a method of any one of the method of making a transgenic plant or alga embodiments herein.

Another embodiment provides a method of elevating cyclic electron transfer (CET) activity in a C3 plant, C4 plant, or alga wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane, a bicarbonate anion transporter, a carbonic anhydrase, a proteorhodopsin protein targeted to thylakoid membranes; and a R carotene monooxygenase protein.

Another embodiment provides a method of enhancing carbon fixation in a C3 plant, C4 plant, or alga wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane, a bicarbonate anion transporter, a carbonic anhydrase, a proteorhodopsin protein targeted to thylakoid membranes; and a R carotene monooxygenase protein.

Another embodiment provides for a method of producing biomass or other products from a C3 plant, C4 plant, or an alga, wherein said products are selected from among starches, oils, fatty acids, triacylglycerols, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane, a bicarbonate anion transporter, a carbonic anhydrase, a proteorhodopsin protein targeted to thylakoid membranes; and a ß carotene monooxygenase protein. The method further comprises growing said plant or alga and harvesting said biomass or recovering said product from said plant or alga.

Another embodiment provides for use of a construct comprising one or more nucleic acids encoding

-   -   a) a PGR5 protein, and a HLA3 protein;     -   b) a PGR5 protein, a HLA3 protein and a PGRL1 protein;     -   c) a PGR5 protein, a HLA3 protein, and a LCIA protein;     -   d) a PGR5 protein, a HLA3 protein, a LCIA protein and a BCA or         HCA2 protein;     -   e) a PGR5 protein, a HLA3 protein, a PGRL1 protein and a LCIA         protein;     -   f) a PGR5 protein, a HLA3 protein, a PGRL1 protein, a LCIA         protein, and a BCA or HCA2 protein;     -   g) a PGR5 protein, a HLA3 protein, and a BCA or HCA2 protein; or     -   h) a PGR5 protein, a HLA3 protein, a PGRL1 protein, and a BCA or         HCA2 protein         for     -   i) making a transgenic plant or alga of a first embodiment;     -   ii) elevating CET activity in a C3 plant, C4 plant, or alga;     -   iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga;         or     -   iv) producing biomass or other products from a C3 plant, C4         plant, or an alga, wherein said products are selected from among         starches, oils, fatty acids, triacylglycerols, lipids, cellulose         or other carbohydrates, alcohols, sugars, nutraceuticals,         pharmaceuticals, fragrance and flavoring compounds, and organic         acids.

Another embodiment provides for use of a construct comprising one or more nucleic acids encoding

-   -   a) a LCIA protein and a BCA or HCA2 protein;         for     -   i) making a transgenic plant or alga of a second embodiment;     -   ii) elevating CET activity in a C3 plant, C4 plant, or alga;     -   iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga;         or     -   iv) producing biomass or other products from a C3 plant, C4         plant, or an alga, wherein said products are selected from among         starches, oils, fatty acids, triacylglycerols, lipids, cellulose         or other carbohydrates, alcohols, sugars, nutraceuticals,         pharmaceuticals, fragrance and flavoring compounds, and organic         acids.

One aspect of the present invention provides for a transgenic plant or alga, comprising within its genome, and expressing or overexpressing, a combination of heterologous nucleotide sequences encoding:

1. i) a PGRL1 protein, a PGR5 protein, and a HLA3 protein; or

-   -   ii) a PGRL1 protein, a PGR5 protein, a HLA3 protein, a LCIA         protein, and a BCA or HCA2 protein; or     -   iii) a Fd1 protein, a HLA3 protein, a LCIA protein, and a BCA or         HCA2 protein; or     -   iv) a leaf FNR1 protein, a HLA3 protein, a LCIA protein, and a         BCA or HCA2 protein; or     -   v) a proteorhodopsin protein targeted to thylakoid membranes, a         HLA3 protein, a LCIA protein, a BCA or HCA2 protein, and a         β-carotene monooxygenase.

2. The transgenic plant or alga of 1, wherein said proteorhodopsin comprises a chloroplast transit peptide selected from among a psbX stop-transfer trans-membrane domain fused to its C-terminus, a DNAJ transit peptide, a CAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide.

3. The transgenic plant or alga of 1 or 2, wherein said β-carotene monooxygenase is expressed under the control of a promoter selected from among an ethanol inducible gene promoter and a green tissue/leaf-specific promoter selected from among CAB and rbcS.

4. The transgenic plant or alga of any one of 1-3, wherein said proteorhodopsin comprises an amino acid substitution selected from among L219E/T206S, M79T, and M79Y, and combinations thereof.

5. The transgenic plant of any one of 1-4, which is a C3 plant or a C4 plant.

6. The transgenic plant of any one of 1-5, which is a transgenic oilseed plant or a transgenic food crop plant.

7. The transgenic oilseed plant of 6, which is selected from among plants of the genera Brassica (e.g., rapeseed/canola (Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea), Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis), coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard other than Arabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm; palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis; Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphea lanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina; Vernonia galamensis; Momordica charantia; and Crambe abyssinica.

8. The transgenic alga of any one of 1-5, which is selected from among Chlorella sp., Nannochloropsis sp., and Chlamydomonas sp.

9. The transgenic plant or alga of any one of 1-8, wherein said heterologous nucleotide sequences are codon-optimized for expression in said transgenic plant or alga.

10. The transgenic plant or alga of any one of 1-9, which exhibits enhanced CO₂ fixation compared to an otherwise identical control plant grown under the same conditions.

11. The transgenic plant or alga of 10, wherein CO₂ fixation is enhanced in the range of from about 10% to about 50% compared to that of an otherwise identical control plant grown under the same conditions.

12. A part of said transgenic plant or alga of any one of 1-11.

13. The part of said transgenic plant of 12, which is selected from among a protoplast, a cell, a tissue, an organ, a cutting, an explant, a reproductive tissue, a vegetative tissue, biomass, an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, a callus, propagation materials, germplasm, cuttings, divisions, and propagations.

14. Progeny or derivatives of said transgenic plant or alga of any one of 1-11.

15. The progeny or derivatives of 14, which is selected from among clones, hybrids, samples, seeds, and harvested material thereof.

16. The progeny of 14 or 15, which is produced sexually.

17. The progeny of 14 or 15, which is produced asexually.

Another aspect of the present invention provides for a method selected from among:

18. i) making a transgenic plant or alga of any one of 1-11;

ii) elevating CET activity in a C3 plant, C4 plant, or alga;

iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga; and

iv) producing biomass or other products from a C3 plant, C4 plant, or alga, wherein said products are selected from among starches, oils, fatty acids, triacylglycerols, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids,

wherein said method comprises expressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, a combination of heterologous nucleotide sequences encoding:

-   -   a) a PGRL1 protein, a PGR5 protein, and a HLA3 protein; or     -   b) a PGRL1 protein, a PGR5 protein, a HLA3 protein, a LCIA         protein, and a BCA or HCA2 protein; or     -   c) a Fd1 protein, a HLA3 protein, a LCIA protein, and a BCA or         HCA2 protein; or     -   d) a leaf FNR1 protein, a HLA3 protein, a LCIA protein, and a         BCA or HCA2 protein; or     -   e) a proteorhodopsin protein targeted to thylakoid membranes, a         HLA3 protein, a LCIA protein, a BCA or HCA2 protein, and a         β-carotene monooxygenase.

19. The method of 18, wherein step iv) further comprises growing said plant or alga and harvesting said biomass or recovering said product from said plant or alga.

20. The method of 18 or 19, wherein said proteorhodopsin comprises a chloroplast transit peptide selected from among a psbX stop-transfer trans-membrane domain fused to its C-terminus, a DNAJ transit peptide, a CAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide.

21. The method of any one of 18-20, wherein said β-carotene monooxygenase is expressed under the control of a promoter selected from among an ethanol inducible gene promoter and a green tissue/leaf-specific promoter selected from among CAB and rbcS.

22. The method of any one of 18-21, wherein said proteorhodopsin comprises an amino acid substitution selected from among L219E/T206S, M79T, and M79Y, and combinations thereof.

23. The method of any one of 18-22, wherein said transgenic plant is a C3 plant, a C4 plant, or an alga.

24. The method of any one of 18-23, wherein said transgenic plant is a transgenic oilseed plant or a transgenic food crop plant.

25. The method of 24, wherein said transgenic oilseed plant is selected from among plants of the genera Brassica (e.g., rapeseed/canola (Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea), Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis), coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard other than Arabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm; palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis; Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphea lanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina; Vernonia galamensis; Momordica charantia; and Crambe abyssinica.

26. The method of any one of 18-23, wherein said alga is selected from among Chlorella sp., Nannochloropsis sp., and Chlamydomonas sp.

27. The method of any one of 18-26, wherein said heterologous nucleotide sequences are codon-optimized for expression in said transgenic plant or alga.

28. The method of any one of 18-27, wherein said transgenic plant or alga exhibits enhanced CO₂ fixation compared to an otherwise identical control plant or alga grown under the same conditions.

29. The method of 28, wherein CO₂ fixation is enhanced in the range of from about 10% to about 50% compared to that of an otherwise identical control plant or alga grown under the same conditions.

Another aspect of the present invention provides for a transgenic plant or alga made by the method of any one of 18-29.

Yet another aspect of the present invention provides for a biomass or other product from a plant or alga, selected from among starches, oils, fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids, made by the method of any one of 18-29.

In addition to the various embodiments listed above, in the Examples below, and in the claims, this disclosure further variously encompasses the presently disclosed and claimed CCM protein combinations in further combinations with the genes and proteins focusing on engineering pathways that are directly involved in facilitating CO₂ transport into leaf chloroplasts, disclosed and claimed in the inventors' previous application PCT International Publication WO 2012/125737. The present disclosure encompasses any combination of genes disclosed herein with any combination of genes disclosed in WO 2012/125737 and in Tables D1-D9 to improve carbon concentrating systems (CCMs) in plants and algae.

Table D1 represents different classes of α-CAs found in mammals.

Table D2-D4 represents representative species, Gene bank accession numbers, and amino acid sequences for various species of suitable CA genes.

Table D5 represents the codon optimized DNA sequence for chloroplast expression in Chlamydomonas reinhardtii. In Table D5, the underlines sequences represent restriction sites, and bases changed to optimize chloroplast expression are listed in lower case. Table D6 provides a breakdown of the number and type of each codon optimized.

Representative species and Gene bank accession numbers for various species of bicarbonate transporter are listed below in Tables D8-D9.

Further scope of the applicability of the presently disclosed embodiments will become apparent from the detailed description and drawing(s) provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of this disclosure, are given by way of illustration only since various changes and modifications within the spirit and scope of these embodiments will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The disclosure can be more fully understood form the following detailed description and the accompanying Sequence Listing, which form a part of this application.

The sequence descriptions summarize the Sequence Listing attached hereto. The Sequence Listing contains standard symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the present disclosure will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawing(s), all of which are given by way of illustration only, and are not limitative of the presently disclosed embodiments, in which:

FIG. 1. Model of the Chlamydomonas CCM showing the localization of inorganic carbon transporters (HLA3, LCIA) and carbonic anhydrase (CAH: CAH1, CAH3, and CAH6) [5]), and Rubisco. LCIB is an essential protein for CCM in Chlamydomonas. It's exact function is unknown.

FIG. 2.(A-B) Growth phenotypes of VVT and HLA3 transgenic (T3) Arabidopsis initially grown on MS media (plus nitrate, NO₃ ⁻). (B) MS media (plus ammonium (NH₄ ⁺) and sucrose) or in soil (ammonium only). X indicates plants died. Numbers refer to plant lines.

FIG. 3.(A-B) Growth phenotypes of VVT and HCA-II transgenic (T1) Arabidopsis 4 weeks after germination. (B) Growth phenotype of VVT Arabidopsis (Col-0, left) and the BCA transgenic (T3) (right).

FIG. 4. Photosynthetic assimilation rate of CO₂ in three transgenic lines (P1, P5, P6) of Arabidopsis expressing BCA (bacterial carbonic anhydrase) measured using a LICOR 6400 gas analyzer. These lines showed ˜30% increase in their photosynthetic efficiency when compared to WT Arabidopsis (Col.-0).

FIG. 5.(A-C) Growth phenotypes of WT and LCIA transgenic (T1) Arabidopsis plants four weeks after germination. (B) Four-week-old WT (left 4 plants) and independent transgenic Camelina (right 4 plants) expressing LCIA. (C) CO₂-dependent photosynthetic rates of WT and LCIA transgenic Camelina.

FIG. 6. Phenotype of HLA3 transgenics grown on nitrate. Energy charge and reductive potential of WT and HLA3 transgenic Arabidopsis. Adenylate, nucleotide cofactors, and inorganic phosphate levels measured as nmole/gFW for plants grown on nitrate. Values are averages±SE.

FIG. 7. Photosynthetically active radiation in proteorhodopsin relative to plant-based chlorophyll [49].

FIG. 8. Plasmid pB110-CAB-PGR5-NOS (Example 1).

FIG. 9. Plasmid pB110-HLA3-pgr5-dsred (Example 1).

FIG. 10. Plasmid pBI 121-CAB1-Tp-NgCAf2-dsred (Example 1).

FIG. 11 illustrates light response curves of Camelina BCA lines.

FIG. 12 illustrates expression of LCIA in Camelina vs WT.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

The following detailed description is provided to aid those skilled in the art in practicing the various embodiments of the present disclosure described herein, including all the methods, uses, compositions, etc., described herein. Even so, the following detailed description should not be construed to unduly limit the present disclosure, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present discoveries.

The present disclosure is explained in greater detail below. This disclosure is not intended to be a detailed catalog of all the different ways in which embodiments of this disclosure can be implemented, or all the features that can be added to the instant embodiments. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which variations and additions do not depart from the scope of the instant disclosure. Hence, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations, and variations thereof.

Any feature, or combination of features, described herein is(are) included within the scope of the present disclosure, provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present disclosure are apparent in the following detailed description and claims.

The contents of all publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety. In case of conflict, the present specification, including explanations of terms, will control.

Definitions

The following definitions are provided to aid the reader in understanding the various aspects of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. Furthermore, the use of the term “including”, as well as other related forms, such as “includes” and “included”, is not limiting.

The term “about” as used herein is a flexible word with a meaning similar to “approximately” or “nearly”. The term “about” indicates that exactitude is not claimed, but rather a contemplated variation. Thus, as used herein, the term “about” means within 1 or 2 standard deviations from the specifically recited value, or ±a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 4%, 3%, 2%, or 1% compared to the specifically recited value.

The term “comprising” as used in a claim herein is open-ended, and means that the claim must have all the features specifically recited therein, but that there is no bar on additional features that are not recited being present as well. The term “comprising” leaves the claim open for the inclusion of unspecified ingredients even in major amounts. The term “consisting essentially of” in a claim means that the invention necessarily includes the listed ingredients, and is open to unlisted ingredients that do not materially affect the basic and novel properties of the invention. A “consisting essentially of” claim occupies a middle ground between closed claims that are written in a closed “consisting of” format and fully open claims that are drafted in a “comprising’ format”. These terms can be used interchangeably herein if, and when, this may become necessary. Furthermore, the use of the term “including”, as well as other related forms, such as “includes” and “included”, is not limiting.

“BCA” refers to bacterial carbonic anhydrase.

“CCMs” and the like refer to carbon concentrating systems.

“CET” refers to cyclic electron transfer.

“LET” refers to linear electron transfer.

“WT” refers to wild-type.

“Cyclic electron transfer modulator protein” refers to any protein natural or synthetic that improves the separation of charge across the thylakoid membrane resulting in improved photophosphorylation with the production of chemical energy. Examples of such modulators are the PGR5 and PRGL1 reductases, however improved proteins in the electron transport chain such as cytochromes, ATPases, ferredoxin-NADP reductase, NAD(P)H-plastoquinone reductase, and the like are also CET modulator proteins.

Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as is known to one of ordinary skill in the art and is understood as included in embodiments where it would be appropriate. Nucleotides may be referred to by their commonly accepted single-letter codes. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUM Biochemical Nomenclature Commission. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description purposes and are not to be unduly limiting.

Regarding disclosed ranges, the endpoints of all ranges directed to the same component or property are inclusive and independently combinable (e.g., ranges of “up to about 25 wt. %, or, more specifically, about 5 wt. % to about 20 wt. %,” is inclusive of the endpoints and all intermediate values of the ranges of “about 5 wt. % to about 25 wt. %,” etc.). Numeric ranges recited with the specification are inclusive of the numbers defining the range and include each integer within the defined range.

As used herein, “altering level of production” or “altering level of expression” means changing, either by increasing or decreasing, the level of production or expression of a nucleic acid sequence or an amino acid sequence (for example a polypeptide, an siRNA, a miRNA, an mRNA, a gene), as compared to a control level of production or expression.

“Conservative amino acid substitutions”: It is well known that certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of biochemical or biological activity. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. Thus, various changes can be made in the amino acid sequences disclosed herein, or in the corresponding DNA sequences that encode these amino acid sequences, without appreciable loss of their biological utility or activity.

Proteins and peptides biologically functionally equivalent to the proteins and peptides disclosed herein include amino acid sequences containing conservative amino acid changes in the fundamental amino acid sequence. In such amino acid sequences, one or more amino acids in the fundamental sequence can be substituted, for example, with another amino acid(s), the charge and polarity of which is similar to that of the native amino acid, i.e., a conservative amino acid substitution, resulting in a silent change.

It should be noted that there are a number of different classification systems in the art that have been developed to describe the interchangeability of amino acids for one another within peptides, polypeptides, and proteins. The following discussion is merely illustrative of some of these systems, and the present disclosure encompasses any of the “conservative” amino acid changes that would be apparent to one of ordinary skill in the art of peptide, polypeptide, and protein chemistry from any of these different systems.

As disclosed in U.S. Pat. No. 5,599,686, certain amino acids in a biologically active peptide, polypeptide, or protein can be replaced by other homologous, isosteric, and/or isoelectronic amino acids, wherein the biological activity of the original molecule is conserved in the modified peptide, polypeptide, or protein. The following list of amino acid replacements is meant to be illustrative and is not limiting:

Original Replacement Amino Acid Amino Acid(s) Ala Gly Arg Lys, ornithine Asn Gln Asp Glu Glu Asp Gln Asn Gly Ala Ile Val, Leu, Met, Nle (norleucine) Leu Ile, Val, Met, Nle Lys Arg Met Leu, Ile, Nle, Val Phe Tyr, Trp Ser Thr Thr Ser Trp Phe, Tyr Tyr Phe, Trp Val Leu, Ile, Met, Nle

In another system, substitutes for an amino acid within a fundamental sequence can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine. and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within a fundamental peptide, polypeptide, or protein sequence can be made by substituting one amino acid within one of these groups with another amino acid within the same group.

Some of the other systems for classifying conservative amino acid interchangeability in peptides, polypeptides, and proteins applicable to the sequences of the present disclosure include, for example, the following:

Functionally defining common properties between individual amino acids by analyzing the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer (1979) Principles of Protein Structure (Springer Advanced Texts in Chemistry), Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on overall protein structure;

Making amino acid changes based on the hydropathic index of amino acids as described by Kyte and Doolittle (1982) J. Mol. Biol. 157(1):105-32. Certain amino acids can be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those that are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred;

Substitution of like amino acids on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in this patent, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Betts and Russell ((2003), “Amino Acid Properties and Consequences of Substitutions”, Bioinformatics for Geneticists, Michael R. Barnes and Ian C. Gray, Eds., John Wiley & Sons, Ltd, Chapter 14, pp. 289-316) review the nature of mutations and the properties of amino acids in a variety of different protein contexts with the purpose of aiding in anticipating and interpreting the effect that a particular amino acid change will have on protein structure and function. The authors point out that features of proteins relevant to considering amino acid mutations include cellular environments, three-dimensional structure, and evolution, as well as the classifications of amino acids based on evolutionary, chemical, and structural principles, and the role for amino acids of different classes in protein structure and function in different contexts. The authors note that classification of amino acids into categories such as those shown in FIG. 14.3 of their review, which involves common physico-chemical properties, size, affinity for water (polar and non-polar; negative or positive charge), aromaticity and aliphaticity, hydrogen-bonding ability, propensity for sharply turning regions, etc., makes it clear that reliance on simple classifications can be dangerous, and suggests that alternative amino acids could be engineered into a protein at each position. Criteria for interpreting how a particular mutation might affect protein structure and function are summarized in section 14.7 of this review, and include first inquiring about the protein, and then about the particular amino acid substitution contemplated.

Biologically/enzymatically functional equivalents of the proteins and peptides disclosed herein can have 10 or fewer conservative amino acid changes, more preferably seven or fewer conservative amino acid changes, and most preferably five or fewer conservative amino acid changes, i.e., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid changes. The encoding nucleotide sequence (e.g., gene, plasmid DNA, cDNA, codon-optimized DNA, or other synthetic DNA) will thus have corresponding base substitutions, permitting it to code for the biologically functionally equivalent form of protein or peptide. Due to the degeneracy of the genetic code, i.e., the existence of more than one codon for most of the amino acids naturally occurring in proteins, other DNA (and RNA) sequences that contain essentially the same genetic information as these nucleic acids, and which encode the same amino acid sequence as that encoded by these nucleic acids, can be used in the methods disclosed herein. This principle applies as well to any of the other nucleotide sequences disclosed herein.

“Control” or “control level” means the level of a molecule, such as a polypeptide or nucleic acid, normally found in nature under a certain condition and/or in a specific genetic background. In certain embodiments, a control level of a molecule can be measured in a cell or specimen that has not been subjected, either directly or indirectly, to a treatment. A control level is also referred to as a wildtype or a basal level. These terms are understood by those of ordinary skill in the art. A control plant, i.e. a plant that does not contain a recombinant DNA that confers (for instance) an enhanced trait in a transgenic plant, is used as a baseline for comparison to identify an enhanced trait in the transgenic plant. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant DNA, or does not contain all of the recombinant DNAs, in the test plant.

The terms “enhance”, “enhanced”, “increase”, or “increased” refer to a statistically significant increase. For the avoidance of doubt, these terms generally refer to about a 5% increase in a given parameter or value, about a 10% increase, about a 15% increase, about a 20% increase, about a 25% increase, about a 30% increase, about a 35% increase, about a 40% increase, about a 45% increase, about a 50% increase, about a 55% increase, about a 60% increase, about a 65% increase, about 70% increase, about a 75% increase, about an 80% increase, about an 85% increase, about a 90% increase, about a 95% increase, about a 100% increase, or more over the control value. These terms also encompass ranges consisting of any lower indicated value to any higher indicated value, for example “from about 5% to about 50%”, etc.

“Expression” or “expressing” refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence. A nucleotide encoding sequence may comprise intervening sequence (e.g., introns) or may lack such intervening non-translated sequences (e.g., as in cDNA). Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated (for example, siRNA, transfer RNA, and ribosomal RNA). The term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors. Thus, expression of a nucleic acid fragment, such as a gene or a promoter region of a gene, may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide), or both.

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively.

The term “genome” as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell. As used herein, the term “genome” refers to the nuclear genome unless indicated otherwise. However, expression in a plastid genome, e.g., a chloroplast genome, or targeting to a plastid genome such as a chloroplast via the use of a plastid targeting sequence, is also encompassed by the present disclosure.

The term “heterologous” refers to a nucleic acid fragment or protein that is foreign to its surroundings. In the context of a nucleic acid fragment, this is typically accomplished by introducing such fragment, derived from one source, into a different host. Heterologous nucleic acid fragments, such as coding sequences that have been inserted into a host organism, are not normally found in the genetic complement of the host organism. As used herein, the term “heterologous” also refers to a nucleic acid fragment derived from the same organism, but which is located in a different, e.g., non-native, location within the genome of this organism. Thus, the organism can have more than the usual number of copy(ies) of such fragment located in its(their) normal position within the genome and in addition, in the case of plant cells, within different genomes within a cell, for example in the nuclear genome and within a plastid or mitochondrial genome as well. A nucleic acid fragment that is heterologous with respect to an organism into which it has been inserted or transferred is sometimes referred to as a “transgene.”

A “heterologous” PGRL1 protein or CAB transit peptide protein-encoding nucleotide sequence, etc., can be one or more additional copies of an endogenous PGRL1 protein or CAB transit peptide protein-encoding nucleotide sequence, or a nucleotide sequence from another plant or other source. PGRL1 is a putative ferredoxin-plastoquinone reductase involved in photosynthetic cyclic electron flow. Furthermore, these can be genomic or non-genomic nucleotide sequences. Non-genomic nucleotide sequences encoding such proteins and peptides include, by way of non-limiting examples, mRNA; synthetically produced DNA including, for example, cDNA and codon-optimized sequences for efficient expression in different transgenic plants algae reflecting the pattern of codon usage in such plants; nucleotide sequences encoding the same proteins or peptides, but which are degenerate in accordance with the degeneracy of the genetic code; which contain conservative amino acid substitutions that do not adversely affect their activity, etc., as known by those of ordinary skill in the art.

The term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members, related sequences, or homologs. The term “homologous” refers to the relationship between two nucleic acid sequence and/or proteins that possess a “common evolutionary origin”, including nucleic acids and/or proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of animal, as well as homologous nucleic acids and/or proteins from different species of animal (for example, myosin light chain polypeptide, etc.; see Reeck et al., (1987) Cell, 50:667). Such proteins (and their encoding nucleic acids) may have sequence homology, as reflected by sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. The methods disclosed herein contemplate the use of the presently disclosed nucleic and protein sequences, as well as sequences having sequence identity and/or similarity, and similar function.

“Host cell” means a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Alternatively, the host cells are monocotyledonous or dicotyledonous plant cells.

The term “introduced” means providing a nucleic acid (e.g., an expression construct) or protein into a cell. “Introduced” includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. “Introduced” includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, can mean “transfection” or “transformation” or “transduction”, and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term “isolated” refers to a material such as a nucleic acid molecule, polypeptide, or small molecule, that has been separated from the environment from which it was obtained. It can also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated. Also intended as “isolated polypeptides” or “isolated nucleic acid molecules”, etc., are polypeptides or nucleic acid molecules that have been purified, partially or substantially, from a recombinant host cell or from a native source.

As used herein, “nucleic acid” or “nucleotide sequence” means a polynucleotide (or oligonucleotide), including single or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases, and unless otherwise indicated, encompasses naturally occurring and synthetic nucleotide analogues having the essential nature of natural nucleotides in that they hybridize to complementary single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. Nucleic acids may also include fragments and modified nucleotide sequences. Nucleic acids disclosed herein can either be naturally occurring, for example genomic nucleic acids, or isolated, purified, non-genomic nucleic acids, including synthetically produced nucleic acid sequences such as those made by solid phase chemical oligonucleotide synthesis, enzymatic synthesis, or by recombinant methods, including for example, cDNA, codon-optimized sequences for efficient expression in different transgenic plants reflecting the pattern of codon usage in such plants, nucleotide sequences that differ from the nucleotide sequences disclosed herein due to the degeneracy of the genetic code but that still encode the protein(s) of interest disclosed herein, nucleotide sequences encoding the presently disclosed protein(s) comprising conservative (or non-conservative) amino acid substitutions that do not adversely affect their normal activity, PCR-amplified nucleotide sequences, and other non-genomic forms of nucleotide sequences familiar to those of ordinary skill in the art.

The protein-encoding nucleotide sequences, and promoter nucleotide sequences used to drive their expression, disclosed herein can be genomic or non-genomic nucleotide sequences. Non-genomic nucleotide protein-encoding sequences and promoters include, for example, naturally-occurring mRNA, synthetically produced mRNA, naturally-occurring DNA, or synthetically produced DNA. Synthetic nucleotide sequences can be produced by means well known in the art, including by chemical or enzymatic synthesis of oligonucleotides, and include, for example, cDNA, codon-optimized sequences for efficient expression in different transgenic plants and algae reflecting the pattern of codon usage in such organisms, variants containing conservative (or non-conservative) amino acid substitutions that do not adversely affect their normal activity, PCR-amplified nucleotide sequences, etc.

“A PGRL1 protein”, “a PGR5 protein”, “a HLA3 protein”, “a CAB transit peptide”, “a PGR5 transit peptide”, or any other protein or peptide presently broadly disclosed and utilized in any of the CCM methods and plants and algae disclosed herein refers to a protein or peptide exhibiting enzymatic/functional activity similar or identical to the enzymatic/functional activity of the specifically named protein or peptide. Enzymatic/functional activities of the proteins and peptides disclosed herein are described below. “Similar” enzymatic/functional activity of a protein or peptide can be in the range of from about 75% to about 125% or more of the enzymatic/functional activity of the specifically named protein or peptide when equal amounts of both proteins or peptides are assayed, tested, or expressed as described below under identical conditions, and can therefore be satisfactorily substituted for the specifically named proteins or peptides in the present enhanced CCM methods and transgenic plants and algae.

“Nucleic acid construct” or “construct” refers to an isolated polynucleotide which can be introduced into a host cell. This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. This construct may comprise an expression cassette that can be introduced into and expressed in a host cell.

“Operably linked” refers to a functional arrangement of elements. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter effects the transcription or expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence.

The terms “plant” or “plants” that can be used in the present methods broadly include the classes of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and unicellular and multicellular algae. The term “plant” also includes plants which have been modified by breeding, mutagenesis, or genetic engineering (transgenic and non-transgenic plants). It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures, seed (including embryo, endosperm, and seed coat) and fruit, plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells, and progeny of same.

Embodiments of the present disclosure also include parts of plants or algae, which can be selected from among a protoplast, a cell, a tissue, an organ, a cutting, an explant, a reproductive tissue, a vegetative tissue, biomass, an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, a callus, propagation materials, germplasm, cuttings, divisions, and propagations.

Other embodiments include progeny or derivatives of transgenic plants and algae disclosed herein selected, for example, from among clones, hybrids, samples, seeds, and harvested material. Progeny can be asexually or sexually produced by methods well known in the art.

Useful C3 and C4 Plants

Plants to which the methods disclosed herein can be advantageously applied include both C3 and C4 plants, including “food crop” and “oilseed” plants, as well as algae.

Food Crop Plants

The term “food crop plant” refers to plants that are either directly edible, or which produce edible products, and that are customarily used to feed humans either directly, or indirectly through animals. Non-limiting examples of such plants include:

1. Cereal crops: wheat, rice, maize (corn), barley, oats, sorghum, rye, and millet;

2. Protein crops: peanuts, chickpeas, lentils, kidney beans, soybeans, lima beans;

3. Roots and tubers: potatoes, sweet potatoes, and cassavas;

4. Oil crops: soybeans, corn, canola, peanuts, palm, coconuts, safflower, cottonseed, sunflower, flax, olive, and safflower;

5. Sugar crops: sugar cane and sugar beets;

6. Fruit crops: bananas, oranges, apples, pears, breadfruit, pineapples, and cherries;

7. Vegetable crops and tubers: tomatoes, lettuce, carrots, melons, asparagus, etc.

8. Nuts: cashews, peanuts, walnuts, pistachio nuts, almonds;

9. Forage and turf grasses;

10. Forage legumes: alfalfa, clover;

11. Drug crops: coffee, cocoa, kola nut, poppy;

12. Spice and flavoring crops: vanilla, sage, thyme, anise, saffron, menthol, peppermint, spearmint, coriander.

In certain embodiments of this disclosure, the food crop plants are soybean, canola, tomato, potato, cassava, wheat, rice, oats, lettuce, broccoli, beets, sugar beets, beans, peas, kale, strawberry, and peanut.

“Oilseed Plants”, “Oil Crop Plants”, “Biofuels Crops”, “Energy Crops”

The terms “oilseed plant” or “oil crop plant”, and the like, to which the present methods and compositions can also be applied, refer to plants that produce seeds or fruit with oil content in the range of from about 1 to 2%, e.g., wheat, to about 20%, e.g., soybeans, to over 40%, e.g., sunflowers and rapeseed (canola). These include major and minor oil crops, as well as wild plant species which are used, or are being investigated and/or developed, as sources of biofuels due to their significant oil production and accumulation.

Exemplary oil seed or oil crop plants useful in practicing the methods disclosed herein include, but are not limited to, plants of the genera Brassica (e.g., rapeseed/canola (Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea), Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis), coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard; wheat; flax (linseed); sunflower; olive; corn; palm; palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis; Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphea lanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina; Vernonia galamensis; Momordica charantia; and Crambe abyssinica.

A non-limiting example of a tuber that accumulates significant amounts of reserve lipids is the tuber of Cyperus esculentus (chufa or tigernuts), which has been proposed as an oil crop for biofuel production. In the case of chufa, use of a constitutive or tuber-specific promoter would be useful in the methods disclosed herein.

Useful Algae

Algae useful in practicing various methods of the present disclosure include members of the following divisions: Chlorophyta and Heterokontophyta.

In certain embodiments, useful algae include members of the following classes: Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certain embodiments, useful algae include members of the following genera: Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, and Ochromonas. In one embodiment, members of the genus Chlorella are preferred.

Some algal species of particular interest include, without limitation: Bacillariophyceae strains, Chlorophyceae, Cyanophyceae, Xanthophyceae, Chrysophyceae, Chlorella, Crypthecodinium, Schizocytrium, Nannochloropsis, Ulkenia, Dunaliella, Cyclotella, Navicula, Nitzschia, Cyclotella, Phaeodactylum, and Thaustochytrid.

Non-limiting examples of algae species that can be used with the methods of the present disclosure include, for example, Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Chlamydomonas moewusii Chlamydomonas reinhardtii Chlamydomonas sp. Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

In certain embodiments of this disclosure, the algae are species of Chlorella, Nannochloropsis, and Chlamydomonas listed above.

Exemplary food crop plant include wheat, rice, maize (corn), barley, oats, sorghum, rye, and millet; peanuts, chickpeas, lentils, kidney beans, soybeans, lima beans; potatoes, sweet potatoes, and cassavas; soybeans, corn, canola, peanuts, palm, coconuts, safflower, cottonseed, sunflower, flax, olive, and safflower; sugar cane and sugar beets; bananas, oranges, apples, pears, breadfruit, pineapples, and cherries; tomatoes, lettuce, carrots, melons, strawberry, asparagus, broccoli, peas, kale, cashews, peanuts, walnuts, pistachio nuts, almonds; forage and turf grasses; alfalfa, clover; coffee, cocoa, kola nut, poppy; vanilla, sage, thyme, anise, saffron, menthol, peppermint, spearmint and coriander and preferably wheat, rice and canola.

The terms “peptide”, “polypeptide”, and “protein” are used to refer to polymers of amino acid residues. These terms are specifically intended to cover naturally occurring biomolecules, as well as those that are recombinantly or synthetically produced, for example by solid phase synthesis.

The term “promoter” or “regulatory element” refers to a region or nucleic acid sequence located upstream or downstream from the start of transcription and which is involved in recognition and binding of RNA polymerase and/or other proteins to initiate transcription of RNA. Promoters need not be of plant or algal origin. For example, promoters derived from plant viruses, such as the CaMV35S promoter, or from other organisms, can be used in variations of the embodiments discussed herein. Promoters useful in the present methods include, for example, constitutive, strong, weak, tissue-specific, cell-type specific, seed-specific, inducible, repressible, and developmentally regulated promoters.

A skilled person appreciates that a promoter sequence can be modified to provide for a range of expression levels of an operably linked heterologous nucleic acid molecule. Less than the entire promoter region can be utilized and the ability to drive expression retained. However, it is recognized that expression levels of mRNA can be decreased with deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. A promoter is classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. It should be understood that the foregoing groups of promoters are non-limiting, and that one skilled in the art could employ other promoters that are not explicitly cited herein.

The term “purified” refers to material such as a nucleic acid, a protein, or a small molecule, which is substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment, and/or which may optionally comprise material not found within the purified material's natural environment. The latter may occur when the material of interest is expressed or synthesized in a non-native environment. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

“Recombinant” refers to a nucleotide sequence, peptide, polypeptide, or protein, expression of which is engineered or manipulated using standard recombinant methodology. This term applies to both the methods and the resulting products. As used herein, a “recombinant construct”, “expression construct”, “chimeric construct”, “construct” and “recombinant expression cassette” are used interchangeably herein.

As used herein, the phrase “sequence identity” or “sequence similarity” is the similarity between two (or more) nucleic acid sequences, or two (or more) amino acid sequences. Sequence identity is frequently measured as the percent of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions.

One of ordinary skill in the art will appreciate that sequence identity ranges are provided for guidance only. It is entirely possible that nucleic acid sequences that do not show a high degree of sequence identity can nevertheless encode amino acid sequences having similar functional activity. It is understood that changes in nucleic acid sequence can be made using the degeneracy of the genetic code to produce multiple nucleic acid molecules that all encode substantially the same protein. Means for making this adjustment are well-known to those of skill in the art. When percentage of sequence identity is used in reference to amino acid sequences it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

Sequence identity (or similarity) can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997)).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; & Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length Win the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17: 191-201 (1993)) low-complexity filters can be employed alone or in combination.

The constructs and methods disclosed herein encompass nucleic acid and protein sequences having sequence identity/sequence similarity at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to those specifically and/or sequences having the same or similar function for example if a protein or nucleic acid is identified with a transit peptide and the transit peptide is cleaved leaving the protein sequence without the transit peptide then the sequence identity/sequence similarity is compared to the protein with and/or without the transit peptide.

A “transgenic” organism, such as a transgenic plant, is a host organism that has been stably or transiently genetically engineered to contain one or more heterologous nucleic acid fragments, including nucleotide coding sequences, expression cassettes, vectors, etc. Introduction of heterologous nucleic acids into a host cell to create a transgenic cell is not limited to any particular mode of delivery, and includes, for example, microinjection, floral dip, adsorption, electroporation, vacuum infiltration, particle gun bombardment, whiskers-mediated transformation, liposome-mediated delivery, Agrobacterium-mediated transfer, the use of viral and retroviral vectors, etc., as is well known to those skilled in the art.

Conventional techniques of molecular biology, recombinant DNA technology, microbiology, and chemistry useful in practicing the methods of the present disclosure are described, for example, in Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press; Ausubel et al. (2003 and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.; Amberg et al. (2005) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition, Cold Spring Harbor Laboratory Press; Roe et al. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee (1990) In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; D. M. J. Lilley and J. E. Dahlberg (1992) Methods in Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA, Academic Press; and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited by Jane Roskams and Linda Rodgers (2002) Cold Spring Harbor Laboratory Press; Burgess and Deutscher (2009) Guide to Protein Purification, Second Edition (Methods in Enzymology, Vol. 463), Academic Press. Note also U.S. Pat. Nos. 8,178,339; 8,119,365; 8,043,842; 8,039,243; 7,303,906; 6,989,265; US20120219994A1; and EP1483367B1. The entire contents of each of these texts and patent documents are herein incorporated by reference.

Preliminary Results: Transgenic Plants Expressing Algal CCM Genes

Previously, reconstitution of a functional inorganic CCM in C3 plants to suppress photo-respiration and enhance photosynthesis was proposed. In WO 2012/125737, it was hypothesized that expression of a minimum of three algal CCM proteins would be sufficient to elevate internal plastid CO₂ concentrations high enough to suppress photorespiration. These three algal CCM genes included the Chlamydomonas plasma membrane-localized and ATP-dependent bicarbonate transporter, HLA3; the chloroplast envelope localized bicarbonate anion transporter, LCIA; and a chloroplast stromal-localized carbonic anhydrase (HCA-II) to accelerate conversion of bicarbonate into CO₂. These genes have individually been shown to be important to the CCM in prior studies ([3-5]). To test this hypothesis, we generated multiple independent transgenic Arabidopsis and Camelina plants expressing each CCM gene as a single gene construct, as well as a stacked 3-gene construct. The expression of each gene was controlled by the light-regulated Cab1 gene promoter [6].

The results of phenotypic analyses of Arabidopsis and Camelina plants transformed with the single CCM gene constructs were as follows:

HLA3 Arabidopsis transgenics varied in their phenotypes, but generally had reduced growth phenotypes relative to wild-type (WT) plants (FIG. 6). When the same plasmid was used to transform Camelina, no viable seeds were recovered from any transformation event after multiple attempts, indicating that HLA3 expression was likely toxic to Camelina.

With respect to carbonic anhydrase (CA) transgenics, we expressed a human carbonic anhydrase-2 (HCA2 (SEQ ID NO:17)) or a bacterial Neisseria gonorrhoeae carbonic anhydrase (BCA SEQ ID NO: 4)) in the chloroplast stroma [7]. We choose these CAs because each has a turnover number (Kcat=106 sec-1) that is approximately 10× faster than plant/algal Cas. In both Arabidopsis and Camelina, we observed phenotypes that were either similar to WT (HCA2) or substantially larger (BCA) than WT plants (FIG. 3B).

Transgenic Arabidopsis plants expressing the LCIA gene were substantially impaired in growth (FIG. 5A). In contrast, Camelina LCIA transgenics grew better than WT, had up to 25% higher photosynthetic rates at ambient CO₂ concentrations, and had reduced CO₂ compensation points (FIG. 5B).

The fact that expression of individual CCM genes impaired growth in C3 plants suggested that additional traits may need to be expressed or silenced to achieve optimal photosynthetic performance.

To determine if we could reconstitute a fully functional CCM complex in C3 plants, we transformed Arabidopsis and Camelina with a triple-gene CCM construct in which the expression of the HLA3, CA, and LCIA genes was driven by the green-tissue specific Cab1 promoter. In both Arabidopsis and Camelina there was either a substantial impairment in growth, or the plants did not survive (results not shown).

Thus, co-expression of the HLA3 gene with any other CCM gene(s) impaired growth even in plants in which expression of the other CCM genes, e.g., LCIA in Camelina, or BCA in Arabidopsis, enhanced growth. These results indicated that HLA3 expression was problematic.

Since the HLA3 protein catalyzes active bicarbonate transport and is the first-dedicated step in the engineered CCM, we re-focused our efforts on trying to determine why HLA3 expression was toxic to plants and how to mitigate its effects. We considered two possible hypotheses for HLA3 toxicity: 1) expression of the HLA3 ABC-transporter increases ATP demand (1 ATP/COO for photosynthesis by 25% and depletes cytoplasmic ATP levels [3-5,8] and 2) elevated bicarbonate levels in HLA3 transgenic plants negatively impact cytoplasmic pH levels. With respect to the latter hypothesis, it is noteworthy that unlike cyanobacteria, plants have robust cytoplasmic CA activity, potentially mitigating the effects of elevated bicarbonate levels on cytoplasmic pH.

The Role of ATP Demand and Cyclic Electron Transfer Activity in CCMs

In contrast to air-grown algae (4 ATP/2 NADPH/CO₂) and C4 plants (5 ATP/2 NADPH/CO₂) which have increased ATP demands for photosynthesis, C3 plants (3 ATP/2 NADPH/CO₂) have limited capacity to generate additional ATP for each electron transferred [8-10]. Increasing ATP demand by 25% per carbon fixed in HLA3 transgenic plants, therefore, could deplete cytoplasmic ATP levels as well as alter the redox state of the cell [8,10]. One mechanism to increase ATP synthesis for each light-driven electron transferred is by cyclic electron transfer (CET) activity. Light-driven CET is catalyzed by photosystem I (PSI) mediated charge separation leading to the reduction of ferredoxin (fd) and the PGR5 protein. The PGR5 protein reduces and protonates plastoquinone (PQ). PQH2 is then oxidized by the cytochrome b6f complex (Cyt b6f). Protons released from the oxidation of PQH2 drive ATP synthesis. The electron transfer cycle is completed by the reduction of plastocyanin (PC) by Cyt b6f, which in turn is oxidized by the PSI primary donor P700+. Significantly, molecular studies have demonstrated that genes encoding proteins functional in CET are substantially overexpressed (4-10×) in C4 plants and air-grown algae relative to related C3 species or high CO₂ grown algae [9,11-17]. These CET genes include: the Proton Gradient Regulation Genes PGR5 and PGRL1, and certain members of the Fd and ferredoxin NADP reductase (FNR) gene families [8-15]: Accession Nos.: PGR5:NM_126585; PGRL1: NM_179091; Fd: AtFd1: At1g10960; AtFd2:At1g60950; FNR: LFNR1:At5g66190; LFRN2: At1g20020) [15]. The sequence for the PRG5 protein with the transit peptide amino acid sequence underlined is provided as

(SEQ ID NO: 1) MAAASISAIG CNQTLIGTSF YGGWGSSISG EDYQTMLSKT VAPPQQARVS RKAIRAVPMMKNVNEGKGLF APLVVVTRNL VGKKRFNQLR GKAIALHSQV ITEFCKSIGA DAKQRQGLIRAKKNGERLG FL. The transit peptide is cleaved to produce the functional PGR5 protein.

To test the hypothesis that ATP depletion in HLA3 transgenics resulted in growth impairment, we compared the phenotypes of VVT and HLA3 transgenics grown on nitrate which would require more linear electron transport (LET) to facilitate the reduction of nitrate. Significantly, the additional ATP produced by LET is not required for conversion of nitrate to ammonium and thus total ATP levels are expected to increase. In contrast, plants grown on ammonium do not require additional LET. Finally, we also grew transgenics on ammonium with sucrose which would presumably provide additional ATP via respiration [15,17]. We hypothesized that growth on nitrate or ammonium with sucrose would provide additional ATP that could potentially drive HLA3 activity.

As shown in FIG. 2B, none of the Arabidopsis HLA3 transgenics (4 independent lines) grew in the presence of ammonium, but all HLA3 lines were rescued when grown on ammonium with sucrose. Furthermore, plants grown on ammonium plus sucrose were phenotypically similar to VVT (FIG. 2B). In contrast, all HLA3 plants grown on nitrate survived, but some lines (#9, #20) had substantially impaired growth phenotypes. Identical results were observed for the germination and growth of VVT and HLA3 transgenic seeds on MS media agar plates using either nitrate (HLA3 transgenics survived) or ammonium (HLA3 transgenics died) as the sole nitrogen source (results not shown). Based on these observations, we propose that increased ATP synthesis associated with nitrate-driven LET and/or sucrose metabolism reduces the depletion of cytoplasmic ATP levels in HLA3 transgenics and rescues them.

This interpretation was corroborated by comparative metabolite analyses of leaf energy charge (EC) status (ATP), inorganic phosphate levels, and leaf reductive potential (RP) of VVT and HLA3 transgenic Arabidopsis grown on nitrate. As shown in FIG. 6, HLA3 transgenics grown on nitrate had reduced EC and RP ratios relative to WT. Energy charge is defined as ([ATP]+½[ADP])/([ATP]+[ADP]+[AMP]). The reduction potential is a measurement of the capacity of the system to gain or lose electrons.

Significantly, inorganic phosphate levels were two-fold higher in HLA3 line #20, while the NADH level was two-fold lower than WT.

These results are consistent with the hypothesis that HLA3 expression places increased ATP demand on plants. This increased ATP demand in HLA3 transgenics may be met in part via NAD(P)H oxidation via the malate/oxaloacetate redox shunt between the mitochondria and chloroplasts [16].

LCIA Phenotype Depends on Plant Species

As previously indicated, LCIA expression in transgenic Arabidopsis resulted in plants with severely depressed growth phenotypes (FIG. 5A). In contrast, transgenic Camelina expressing LCIA had increased growth rates as well as higher CO₂-dependent photosynthetic rates relative to WT (FIG. 5B). We propose that the substantially greater carbon sink-strength of Camelina relative to Arabidopsis accounts for the enhanced growth phenotype observed in Camelina LCIA plants. In support of this hypothesis, we observed that Camelina LCIA transgenics had higher CO₂-dependent rates of photosynthesis and lower CO₂ compensation points (40 vs. 53 ppm CO₂) than WT plants indicative of facilitated inorganic carbon uptake by LCIA (FIG. 5C).

Overview: Enhancing photosynthetic carbon fixation by increasing ATP production and limiting CO₂ diffusion out of artificial CCM lines; Strategies for facilitating CET and ATP synthesis in C3 plants

Prior attempts to subvert the limitations of photosynthesis have focused on engineering RuBisCO throughput and specificity [35] by introduction of engineered and non-native forms of the enzyme [36], through alterations in the regenerative capacity of the Calvin cycle [37,38] or by engineering photorespiratory bypasses [39]. These studies produced mixed results, thus advocating for a more comprehensive systems-level approach to enhance and/or redirect photosynthetic carbon flux.

As evidenced by our prior work described above, we postulate that both the carbon assimilatory steps and the light-based generation of ATP and NAPDH must be considered to develop a competent CCM with significantly improved photosynthetic capacity. To demonstrate proof of concept, an Arabidopsis line that contains a functional CCM that includes mechanisms to adjust ATP levels to meet transporter demand will be generated.

Enhancing CET and ATP Synthesis to Support HLA3-Dependent Bicarbonate Uptake

To exploit the expression of an algal CCM in C3 plants requires that we meet the additional energy demands required to actively transport inorganic carbon. As previously discussed in the section entitled “The role of ATP demand and cyclic electron transfer activity in CCMs”, C4 plants and algae have robust CET activity, and overexpress a variety of genes involved in CET [13,16,40-45] compared to C3 plants.

Several strategies are identified in the following examples, to increase ATP synthesis to support HLA3-dependent bicarbonate transport. Several of these strategies focus on elevating CET activity in C3 plants. Another approach involves the expression of a green photon-driven bacterial proton pump in thylakoids to supplement proton-driven ATP synthesis. Each approach is designed to complement existing CCM lines in Arabidopsis, Camelina, and potato we have created, and are evaluated based upon measured adenylate levels, plant biomass production, and photosynthetic measurements of carbon assimilation. The materials and methods employed in the examples below are for illustrative purposes only, and are not intended to limit the practice of the present embodiments thereto. Any materials and methods similar or equivalent to those described herein as would be apparent to one of ordinary skill in the art can be used in the testing or practice of the present embodiments, i.e., the materials, methods, and examples are illustrative only and not intended to be limiting.

Example 1: Enhancing CET Based on Overexpressing the Proton Gradient Regulatory Proteins PGR5 and PGRL1 in C3 Plants

Enhancing CET is based on overexpressing the proton gradient regulatory proteins PGR5 and/or PGRL1 which have previously been shown to be important to CET [37].

It has recently been demonstrated that the PGRL1 protein has antimycin A-sensitive (AA), ferredoxin-plastoquinone reductase (FQR) activity [46]. In Chlamydomonas, PGRL1 is part of the Cytb6f/PSI supercomplex which mediates CET. Significantly, PGRL1 forms homodimers as well as heterodimers with PGR5 via redox active cysteine residues. Under high-light conditions, thioredoxinred reduces PGRL1 dimers present in grana stacks, increasing the abundance of PGRL1 monomers and enhancing CET [47]. Mutational studies have shown that the PGR5 protein is required for Fd oxidation and PGRL1 reduction, but not for PQ reduction. In addition, it has been shown that PGRL1/PGR5 heterodimers are more active in CET than PGRL1 monomers. In C4 plants PGR5 and PGRL1 expression levels are elevated (4×) relative to C3 plants [9]. Similarly, PGR5 expression is up-regulated in air-grown Chlamydomonas (active CCM and HLA3 activity) relative to high CO₂ (low CCM) grown cells [16,43]. Significantly, overexpression of PGRL1 and PGR5 has also been shown to increase AA-sensitive CET in transgenic Arabidopsis [48]. One embodiment of the present invention provides for an overexpression of PGRL1 gene (SEQ ID NO:106) and PGR5 gene with chloroplast targeting sequence (SEQ ID NO:2) with HLA3 gene (SEQ ID NO:12) or with HLA3 gene (SEQ ID NO:12) and LCIA gene (SEQ ID NO:16) and BCA gene codon optimized for expression in Arabidopsis (SEQ ID NO:4) to yield substantially increased photosynthetic rates, particularly in plants with enhanced sink strength (Camelina and potato for example). Co-expression of the PGR5 gene (SEQ ID NO:2) along with the HLA3 gene (SEQ ID NO:12) in Camelina rescued the HLA3 gene and it was no longer lethal. These results indicate that the PGR5 gene is enabling the production of sufficient ATP to meet the demands of the HLA3 gene product.

HLA3 (SEQ ID NO:12) and PGR5 (SEQ ID NO:2) are introduced as a double construct into Arabidopsis or Camelina, by Agrobacterium-mediated Ti plasmid transformation using, for example, plasmid pB110-HLA3-pgr5-dsred (FIG. 9). Since PGR5 protein (SEQ ID NO:1) is naturally targeted to the thylakoid membranes, no additional targeting sequences are introduced. Similarly, since HLA3 protein (SEQ ID NO:77) is naturally targeted to the chloroplast envelope, no additional targeting sequences are added. HLA3 is codon optimized for plant expression.

In one embodiment, the expression of each protein is driven by the light sensitive leaf-specific CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9) (FIG. 9).

The BCA gene (AAW89307; SEQ ID NO:4), under the control of CAB1 promoter, is introduced in to Arabidopsis by Agrobacterium-mediated Ti plasmid transformation by floral dip method using the construct shown in FIG. 10.

As a visual marker, the plasmid also includes a gene for expression of fluorescent DsRed protein under the control of CVMV promoter and Nos terminator (FIG. 10).

Plants are transformed by vacuum infiltration method (Lu and Kang (February, 2008) Plant Cell Rep. 27(2):273-8), and will be screened for biomass yield parameters (including plant weight, height, branching and seed yield) and photosynthetic efficiency measured as CO₂ absorption with the aid of a LiCor 6400 gas exchange analyzer.

The PGRL1 gene from Arabidopsis (NM_179091 SEQ ID NO:3) will be subcloned into pCambia1301-based binary plasmid under control of the CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9). The plasmid will also carry a gene for hygromycin selection marker. Agrobacterium-mediated transformation takes place by the standard floral dip method followed by germination of seeds on hygromycin to select for transformants. The expression of PRGL1 will be confirmed by RT-PCR, and the resulting transgenic plant lines will be crossed with HLA3/PGR5 plants and screened for biomass yield and photosynthesis rate (CO₂ fixation).

Example 2: Determining if Fd1 Gene Overexpression can Support Algal CCM and Increased Photosynthetic Rates

It has recently been demonstrated that specific members of the ferredoxin (Fd) gene family facilitate CET. Overexpression of pea ferredoxin1 (Fd1) enhanced CET at the expense of LET in tobacco [16,40].

Therefore, another embodiment of the present invention provides enhancing ATP production and titrating the expression of the pea Fd1 gene in the three model C3 plants with and without co-expression of the CCM genes to determine if Fd1 overexpression can support the algal CCM and increased photosynthetic rates. Earlier results demonstrated that Fd1 overexpression slightly impaired Linear Electron Transfer (LET), resulting in a stunted phenotype [40]. We expect that the additional ATP demand in HLA3 transgenics, however, will mitigate these effects.

Fd1 gene (At1g10960) will be introduced by Agrobacterium-mediated Ti plasmid transformation. Fd1 gene will be subcloned into pCambia1301-based binary plasmid under control of CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9). The plasmid will also carry a gene for hygromycin selection as a marker. Agrobacterium-mediated transformation takes place by the standard floral dip method, followed by germination of seeds on hygromycin to select for transformants. The expression of FD1 (SEQ ID NO:93) will be confirmed by real time QPCR, and the resulting plant lines exhibiting different levels of FD1 expression will be crossed with CCM-expressing plants and screened for biomass yield and photosynthesis rate with the aid of a LiCor 6400 CO₂-gas exchange analyzer.

Example 3: Overexpression of Unique Ferredoxin NADP Reductase (FNR) Gene Family Members Associated with CET

Yet another embodiment is based on overexpression of unique ferredoxin NADP reductase (FNR) gene family members associated with CET. Leaf FNR (LFNR) catalyzes the reduction of Fd and is involved in both LET and CET [15]. It was recently demonstrated that there are three LFNR gene family members expressed in maize leaves: Accession Nos. BAA88236 (LFNR1), BAA88237 (LFNR2), and ACF85815 (LFNR3).

LFNR-1 was shown to be localized to thylakoid membranes and associated with Cytb6f complexes. LFNR2 was present in thylakoids and stroma associated with Cytb6f complexes. LFNR3 was soluble and not associated with Cytb6f complexes.

Significantly, when plants were grown with nitrate instead of ammonium, expression of LFNR1 and LFNR2 was elevated but not that of LFNR3. In contrast, studies using Arabidopsis LFNR1 knock out mutants demonstrated that PGA-dependent oxygen evolution (which requires additional ATP) is more negatively affected than is nitrate-dependent oxygen evolution (no additional ATP demand), suggesting that LFNR1 may play a role in regulating CET [15]. However, this interpretation remains equivocal.

To determine if CET activity and HLA3 mediated inorganic carbon uptake can be altered by differential expression of LFNR1, we will both over-express (CAB1 promoter (SEQ ID NO:7)) and under-express (LFNR1 RNAi) LFNR1 in transgenic Arabidopsis to determine the impact of altered LFNR1 expression on functional CCM activity.

For overexpression of the LFNR1, the gene (At5g66190) will be introduced by Agrobacterium-mediated Ti plasmid transformation by floral dipping. The LFNR1gene will be subcloned into pCambia1301-based binary plasmid under control of the CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9). The plasmid will also carry a gene for hygromycin selection as a marker. The expression of LFNR1 will be confirmed by real time QPCR, the resulting plant lines will be crossed with CCM-expressing plants, and screened for biomass yield and photosynthesis rate with the aid of a LiCor 6400 CO₂-gas exchange analyzer.

For downregulaton of the LFNR1 levels, an RNAi construct containing a partial sequence of the LFNR1 (At5g66190 or BAA88236) and reverse complementary sequence of LFNR1 will be subcloned into pCambia1301-based binary plasmid under control of the CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9). The plasmid will also carry a gene for hygromycin selection as a marker. The reduced level of LFNR1 expression will be confirmed by real time QPCR.

The resulting lines will be crossed with CCM-expressing lines to generate double mutants. Those mutants will be screened for biomass yield parameters (including plant weight, height, branching and seed yield) and photosynthetic efficiency measured as CO₂ absorption with the aid of a LiCor 6400 gas exchange analyzer.

Example 4: Facilitated Vectoral Proton Transport Using Proteorhodopsin (PR)

In yet another embodiment green photons, not absorbed by chlorophyll, to drive proton transport across thylakoids by expressing modified PR [49]) will be employed to enhance ATP synthesis (FIG. 7).

PR is a seven-helix transmembrane-spanning protein similar to bacteriorhodopsin that contains retinal in its active site. Green light-driven cis-trans isomerization of retinal drives vectoral proton transfer across the membrane [50-55]. Significantly, it has been demonstrated that a functional PR could be expressed in a respiration-impaired mutant of E. coli when supplemented with exogenous all-trans retinal [56]. More recently, hydrogen production was shown to increase nearly two-fold in PR-expressing E. coli when cells were exposed to increasing light intensities (70 to 130 μE), indicating that PR can efficiently absorb light even at low intensities [57]. To the best of our knowledge, retinal complementation of other rhodopsins has not been reported. Significantly, PR-expressing E. coli respiratory mutants generated sufficient proton-motive force to support ATP synthesis levels, leading to enhanced cell viability and motility when transgenics were exposed to sunlight as the only energy source.

These results suggest that targeting PR to the thylakoid membrane using appropriate targeting sequences (e.g., nuclear-encoded, N-terminal, light harvesting complex signal sequences) and supplementation with exogenous retinal or retinal derived from β-carotene cleavage) could drive additional ATP synthesis. One concern is that the optical cross section of retinal is small and light harvesting by PR is not supplemented by antenna complexes. This constraint may be overcome in part by overexpressing PR in thylakoids. Regardless, the additional proton gradient necessary to support HLA3 activity is substantially less than that required to support overall CO₂ fixation. The best achievable PR expression levels will be determined empirically using different gene promoters, e.g., psaD (SEQ ID NO:10), rbcs (SEQ ID NO:11), and cab1 (SEQ ID NO:7), to drive its expression.

Generation of Improved PR and its Functional Reconstitution in Chloroplasts

PR (AF279106), for example (SEQ ID NO:98), will be introduced into Arabidopsis, Camelina, and potato by Ti plasmid transformation and targeted to the thylakoid membrane using the DNAJ transit peptide (At5g21430, SEQ ID NO: 22) or psbX stop-transfer trans-membrane domain (At2g06520 SEQ ID NO:23) fused to the C-terminus of PR [58], or transit peptides from nuclear encoded chloroplast proteins such as CAB (SEQ ID NO:13), PGR5 (SEQ ID NO:14), and psaD (SEQ ID NO:15). Reconstitution with exogenous retinal will be carried out in a manner similar to strategies described for E. coli, except that retinal will be painted on the surface of the leaf [56] to demonstrate proof of concept. Retinal reconstitution will be followed by monitoring the absorption of the thylakoid membranes at 540 nm [59].

If exogenously applied retinal is not incorporated into PR, we will express low levels of a plant codon-optimized β-carotene monooxygenase for example (SEQ ID NO:100) in plastids to cleave a small fraction of β-carotene to generate retinal. Non-limiting examples of β-carotene monooxygenases that can be used include, for example, mouse, human, zebra fish, and rat enzymes (Accession Nos. AW044715, AK001592, AJ290390, and NM_053648, respectively). Alternatively, if β-carotene levels are severely depleted, we will transiently express β-carotene monooxygenase under the control of a transient inducible promoter such as an ethanol inducible gene promoter. This is available as an EcoRl/Pstl fragment from Syngenta-Construct: pJL67-5S::AlcR/AlcA::GUS in pMLBART (Weigel World, Max Planck Institute for Developmental Biology, Tubingen, Germany) for periods of time sufficient to fully saturate PR [60,61]. Operation of a functional retinal photocycle in PR will be confirmed by transient absorption spectroscopy [62].

Alternatively, promoters such as the green tissue/leaf-specific promoters such as the CAB (At3g54890 SEQ ID NO:7) and rbcS (At5g38420 SEQ ID NO:11) promoters can be used, for example see SEQ ID NO:5 for the BCA protein with a rbc-1a transit peptide. As the skilled person will be well aware, various promoters may be used to promote the transcription of the nucleic acid of the invention, i.e. the nucleic acid which when transcribed yields an RNA molecule that modulates the expression and/or activity of a protein according to the invention. Such promoters include for example constitutive promoters, inducible promoters (e.g. light inducible promoters, stress-inducible promoters, drought-inducible promoters, hormone-inducible promoters, chemical-inducible promoters, etc.), tissue-specific promoters, developmentally regulated promoters and the like.

Thus, a plant expressible promoter can be a constitutive promoter, i.e. a promoter capable of directing high levels of expression in most cell types (in a spatio-temporal independent manner). Examples of plant expressible constitutive promoters include promoters of bacterial origin, such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, but also promoters of viral origin, such as that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al., 1988, Mol. Gen. Genet. 212: 182-190) or 19S RNAs genes (Odell et al., 1985, Nature. 6; 313(6005):810-2; U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al., 1989, EMBO J. 8:2195-2202), the enhanced 2×35S promoter (Kay at al., 1987, Science 236:1299-1302; Datla et al. (1993), Plant Sci 94:139-149) promoters of the cassava vein mosaic virus (CsVMV; WO 97/48819, U.S. Pat. No. 7,053,205), 2×CsVMV (WO2004/053135) the circovirus (AU 689 311) promoter, the sugarcane bacilliform badnavirus (ScBV) promoter (Samac et al., 2004, Transgenic Res. 13(4):349-61), the figwort mosaic virus (FMV) promoter (Sanger et al., 1990, Plant Mol Biol. 14(3):433-43), the subterranean clover virus promoter No 4 or No 7 (WO 96/06932) and the enhanced 35S promoter as described in U.S. Pat. Nos. 5,164,316, 5,196,525, 5,322,938, 5,359,142 and 5,424,200. Among the promoters of plant origin, mention will be made of the promoters of the promoter of the Arabidopsis thaliana histone H4 gene (Chabouté et al., 1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol. 29:637-649, U.S. Pat. No. 5,510,474) of Maize, Rice and sugarcane, the Rice actin 1 promoter (Act-1, U.S. Pat. No. 5,641,876), the histone promoters as described in EP 0 507 698 A1, the Maize alcohol dehydrogenase 1 promoter (Adh-1) (from the world wide web at patentlens.net/daisy/promoters/242.html)).

A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to heat, cold, drought, light etc.), timing, developmental stage, and the like.

Promoters that can be used to practice this invention include those that are green tissue specific such as the promoter of light harvesting complex protein 2 (Sakamoto et al. Plant Cell Physiology, 1991, 32(3): 385-393) or the promoter of the cytosolic fructose-1, 6-bisphosphatase from rice (Si et al. Acta Botanica Sinica 45: 3(2003): 359-364). Alternative embodiments include light inducible promoters such as promoters of the plant ribulose-biscarboxylase/oxygenase (Rubisco) small subunit promoter (U.S. Pat. No. 4,962,028; WO99/25842) from Zea mays and sunflower. Also the small subunit promoter from Chrysanthemum may be used, combined or not combined with the use of the respective terminator (Outchkourov et al., Planta, 216: 1003-1012, 2003).

Additional promoters that can be used to practice this invention are those that elicit expression in response to stresses, such as the RD29 promoters that are activated in response to drought, low temperature, salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, Plant Cell, Vol. 6, 251-264; WO12/101118), but also promoters that are induced in response to heat (e.g., see Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1: 471-478, and the maize rbcS promoter, Schaffher and Sheen (1991) Plant Cell 3: 997-1012); wounding (e.g., wunl, Siebertz et al. (1989) Plant Cell 1: 961-968); pathogens (such as the PR-I promoter described in Buchel et al. (1999) Plant Mol. Biol. 40: 387-396, and the PDF 1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (e.g., see Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447-458).

Use may also be made of salt-inducible promoters such as the salt-inducible NHX1 promoter of rice landrace Pokkali (PKN) (Jahan et al., 6^(th) International Rice Genetics symposium, 2009, poster abstract P4-37), the salt inducible promoter of the vacuolar H+-pyrophosphatase from Thellungiella halophila (TsVP1) (Sun et al., BMC Plant Biology 2010, 10:90), the salt-inducible promoter of the Citrus sinensis gene encoding phospholipid hydroperoxide isoform gpx1 (Avsian-Kretchmer et al., Plant Physiology July 2004 vol. 135, p 1685-1696).

In alternative embodiments, tissue-specific and/or developmental stage-specific promoters are used, e.g., promoter that can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77, describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter eIF4. Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used. Other promoters that can be used to express the nucleic acids of the invention include, a leaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285 1295, describing a leaf-specific promoter in maize); a tomato promoter active during fruit ripening, senescence and abscission of leaves, a guard-cell preferential promoter e.g. as described in PCT/EP12/065608, and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); the Blec4 gene from pea, which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers; the ovule-specific BELI gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells. Further tissue specific promoters that may be used according to the invention include, promoters active in vascular tissue (e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), carpels (e.g., see Ohl et al. (1990) Plant Cell 2. In alternative embodiments, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids used to practice the invention. For example, the invention can use the auxin-response elements EI promoter fragment (AuxREs) in the soybean {Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (ABA) (Sheen (1996) Science 274:1900-1902). Further hormone inducible promoters that may be used include auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 11: 323-334), cytokinin-inducible promoter (e.g., see Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters responsive to gibberellin (e.g., see Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825) and the like.

In alternative embodiments, nucleic acids used to practice the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324). Using chemically- {e.g., hormone- or pesticide) induced promoters, i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field, expression of a polypeptide of the invention can be induced at a particular stage of development of the plant. Use may also be made of the estrogen-inducible expression system as described in U.S. Pat. No. 6,784,340 and Zuo et al. (2000, Plant J. 24: 265-273) to drive the expression of the nucleic acids used to practice the invention.

In alternative embodiments, a promoter may be used whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.

In alternative embodiments, a tissue-specific plant promoter may drive expression of operably linked sequences in tissues other than the target tissue. In alternative embodiments, a tissue-specific promoter that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well, is used.

According to the invention, use may also be made, in combination with the promoter, of other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators (“enhancers”), for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example.

Other regulatory sequences that enhance the expression of the nucleic acid of the invention may also be located within the chimeric gene. One example of such regulatory sequences is introns. Introns are intervening sequences present in the pre-mRNA but absent in the mature RNA following excision by a precise splicing mechanism. The ability of natural introns to enhance gene expression, a process referred to as intron-mediated enhancement (IME), has been known in various organisms, including mammals, insects, nematodes and plants (WO 07/098042, p 11-12). IME is generally described as a posttranscriptional mechanism leading to increased gene expression by stabilization of the transcript. The intron is required to be positioned between the promoter and the coding sequence in the normal orientation. However, some introns have also been described to affect translation, to function as promoters or as position and orientation independent transcriptional enhancers (Chaubet-Gigot et al., 2001, Plant Mol Biol. 45(1):17-30, p 27-28).

Examples of genes containing such introns include the 5′ introns from the rice actin 1 gene (see U.S. Pat. No. 5,641,876), the rice actin 2 gene, the maize sucrose synthase gene (Clancy and Hannah, 2002, Plant Physiol. 130(2):918-29), the maize alcohol dehydrogenase-1 (Adh-1) and Bronze-1 genes (Callis et al. 1987 Genes Dev. 1(10):1183-200; Mascarenhas et al. 1990, Plant Mol Biol. 15(6):913-20), the maize heat shock protein 70 gene (see U.S. Pat. No. 5,593,874), the maize shrunken 1 gene, the light sensitive 1 gene of Solanum tuberosum, and the heat shock protein 70 gene of Petunia hybrida (see U.S. Pat. No. 5,659,122), the replacement histone H3 gene from alfalfa (Keleman et al. 2002 Transgenic Res. 11(1):69-72) and either replacement histone H3 (histone H3.3-like) gene of Arabidopsis thaliana (Chaubet-Gigot et al., 2001, Plant Mol Biol. 45(1):17-30).

Other suitable regulatory sequences include 5′ UTRs. As used herein, a 5′ UTR, also referred to as a leader sequence, is a particular region of a messenger RNA (mRNA) located between the transcription start site and the start codon of the coding region. It is involved in mRNA stability and translation efficiency. For example, the 5′ untranslated leader of a petunia chlorophyll a/b binding protein gene downstream of the 35S transcription start site can be utilized to augment steady-state levels of reporter gene expression (Harpster et al., 1988, Mol Gen Genet. 212(1):182-90). WO95/006742 describes the use of 5′ non-translated leader sequences derived from genes coding for heat shock proteins to increase transgene expression.

The chimeric gene may also comprise a 3′ end region, i.e. a transcription termination or polyadenylation sequence, operable in plant cells. As a transcription termination or polyadenylation sequence, use may be made of any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV 35S terminator, or of plant origin, such as for example a histone terminator as described in published Patent Application EP 0 633 317 A1. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

The expression and targeting of proteorhodopsin to the thylakoid membranes will take advantage of the green energy spectrum that is inaccessible to chlorophyll. An increase in the amount of ATP is expected under photosynthesis conditions, from proton gradient generated both by the photosystems and the proteorhodopsin pump. Under conditions of inhibition of electron transfer through the photosystems, we should be able to observe a steady rate of ATP synthesis well above the basal rate through the activity of the proteorhodopsin proton pump.

Under normal pH conditions, protons are pumped into the bacterial periplasmic space by PR [50]. The photo-driven retinal cycle begins with photoisomerization of all trans-retinal to 13-cis retinal. The resulting conformational change poises the system for transfer of a proton from the Schiff base (SB; pKa˜11) to the counter ion, Asp 97 (pKa˜7.5). The proton is transferred to the lumen via a proton-conducting channel, and the SB is reprotonated from the cytoplasm. The mechanism of proton release in PR is not as well understood as in bacteriorhodopsin (BR); however, the main events of the photocycle are expected to be similar to those of BR. One potential challenge for pumping protons by PR in thylakoid membranes is the pH gradient-dependent reversibility of proton transfer by PR. At periplasmic pHs, <5.5, proton flow in PR is reversed, potentially depleting the proton gradient and impairing ATP synthesis. Thus, at the lumenal pH of thylakoids (4.5), reversed proton transduction via PR is possible. One of the critical residues involved in reversible proton flow is Asp97, which acts as the proton acceptor from retinal. The pKa of Asp97 in PR is ˜7.5, while the pKa of its counterpart in BR is ˜2.5. Due to the extremely low pKa of the counter ion, BR is able to retain its forward pumping activity at pHs as low as 3.5. The ability of PR to act as a proton pump in the thylakoid membrane thus entails maintaining the pumping efficiency at low pH conditions prevailing in the lumen. We propose that vectoral pumping of protons into the thylakoid lumen can be achieved by lowering the pKa of Asp97 and/or by protecting the SB from the lumenal pH through rational, site-specific mutagenesis. The electrostatic environment around the SB in PR is presumably maintained by the counter ions, Asp97, Asp227 (analogous to BR Asp212), Arg94 (analogous to BR Arg82) and His75. In BR, the low pKa of Asp85 is attributed to its strong hydrogen bonding interactions with Thr89 and Arg82 [53,54]. Since, interactions that reduce the pKa of Asp97 will promote proton-pumping activity at low external pH, mutation of Met79 to a residue that can hydrogen bond to His75 and Asp212, like Tyr or Thr, will be explored. These mutations are proposed by overlaying the structures of BR and PR, and identifying residues which are in a position to effect the desired behavior. Finally, the ability of a modified PR to work as an efficient H+ pump at acidic pHs will also entail shielding the SB from the extracellular environment. To this end, a L219E/T206S mutant will be generated, wherein E219 and S206 will form a Glu-Ser gate regulating vectoral proton transfer as occurs in BR.

To determine if any transgenes alter CET or ATP synthesis activity, we will compare the dark reduction kinetics of the photosystem I primary donor, P700+ in VVT and transgenic plants, with and without dibromothymoquinone (DBMIB), an inhibitor of Cytb6f-mediated CET. Dark P700+ reduction kinetics are expected to be faster in plants with more active CET. In addition, we will assess the amplitude of the After Glow (AG) thermoluminescence band (−40° C.) associated with CET activity [11,14,16,43,63]. Pool sizes of ATP will also be assessed in VVT and transgenic plants by mass spectroscopy.

Referring now to FIG. 11, additional transgenic Camelina lines were produced that expressed the BCA gene (SEQ ID NO:4) in the chloroplast stroma. These lines were produced using the Agrobacterium-mediated transformation procedures as described previously. Three lines were evaluated for their ability to accumulate biomass and provide improved photosynthetic rates. Wildtype Camelina and the BCA mutant lines were not significantly different at lower light levels (0-400 umol/m²/s) in their ability to assimilate carbon dioxide. However, as light intensity increased the BCA transformants showed between 10 and 30% higher accumulation of CO₂ at 2000 μmoles/m²/s than wildtype. The BCA line 9.2 was the highest while lines BCA 4.1 and BCA 5.7 were both about 10% higher than wildtype. This improved ability to assimilate CO₂ was reflected in two of the lines (BCA-5.7 and BCA-9.2) into increased biomass accumulation, with these lines having about 15% greater biomass accumulation than wildtype. The BCA-4.1 line did not show improved biomass accumulation compared to control.

Referring now to FIG. 12, the ability of the chloroplast enveloped localized bicarbonate transporter bicarbonate transporter (LCIA) protein to transport bicarbonate and improve the capture of inorganic carbon by transgenic Camelina was determined following the method of Farquhar and colleagues (1989). LCIA transgenic Camelina were produced using the Agrobacterium-mediated transformation processed described previously. A LCIA expressing mutant line (CAM-LCIA) was compared to wildtype Camelina (Cam-WT) for the observed discrimination of the stable isotope ¹³C. This carbon isotope discrimination is expressed as the difference between the ¹³C in the air and in a plant which has been previously exposed to ¹³CO₂, the carbon isotope discrimination is symbolized by A and expressed in parts per million (ppm) and is described by Farquhar and colleagues (1989). In the LCIA transgenic lines, the observed discrimination by the plant was 20% less than that observed in the wildtype. This indicates that the insertion of LCIA provides the plant the ability to better accumulate and retain inorganic carbon than the wildtype plant and shows decreased “leakiness” vs wildtype. Reference for ¹³C discrimination: Carbon isotope discrimination and photosynthesis, G. D. Farquhar, J. R. Ehlieringer and K. T. Hubick. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989, 40, 503-537.

TABLE D1 Kcat/ Kcat Km Km Ki Subcellular Tissue I organ Isoenzyme (s−1) (mM) (M⁻¹s⁻¹) (nM) localization localization hCAI  2 × 10⁵ 4.0 5.0 × 10⁷ 250 cytosol E, GI hCAII  1.4 × 10^(6o) 9.3 1.5 × 10⁸ 12 cytosol E, eye, GI, BO, K, L, T, B hCAIII 1.0 × 10⁴ 33.3 3.0 × 10⁵ 2 × 10⁵ cytosol SM, A hCAIV 1.0 × 10⁶ 21.5 5.1 × 10⁷ 74 membrane K, L, P, B, C, H hCAVA 2.9 × 10⁵ 10.0 2.9 × 10⁷ 63 mitochondria Li hCAVB 9.5 × 10⁵ 9.7 9.8 × 10⁷ 54 mitochondria H, SM, P, K, SC, GI hCAVI 3.4 × 10⁵ 6.9 4.9 × 10⁷ 11 secreted G hCAVII 9.5 × 10⁵ 11.4 8.3 × 10¹ 2.5 cytosol CNS hCAVIII cytosol CNS hCAIX 3.8 × 10⁵ 6.9 5.5 × 10⁷ 25 transmembrane TU, GI hCAX cytosol CNS hCAXI cytosol CNS hCAXII 4.2 × 10⁵ 12.0 3.5 × 10⁷ 5.7 transmembrane R, I, RE, eye, TU hCAXIII 1.5 × 10⁵ 13.8 1.1 × 10⁷ 16 cytosol K, B, L, GI, RE hCAXIV 3.1 × 10⁵ 7.9 3.9 × 10⁷ 41 transmembrane K, B, L hCAXV 4.7 × 10⁵ 14.2 3.3 × 10⁷ 72 membrane K H = Human; M = Mouse; hCAVIII, X, and XI are devoid of catalytic activity. E = Erthrocyes; GI = GI tract; BO = Bone osteoclasts; K = kidney, L = Lung; T = testis; B = brain; SM = skeletal muscle; A = Adipocytes; P = pancreas; C = colon; H = heart; Li = liver; SC = spinal cord; G = salivary and mammary gland; R = renal; I = intestinal; TU = tumors, RE = Reproductive

TABLE D2 Exemplary Type II Carbonic Anhydrases Accession Organism Sequence Number SEQ. ID. NO Human MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY NP-000058.1 SEQ. ID. NO. 19 DPSLKPLSVS YDQATSLRIL NNGHAFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QVLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK Macaca MSHHWGYGKH NGPEHWHKDF PIAKGQRQSP VDIDTHTAKY BAE91302.1 SEQ. ID. NO. 24 fascicularis DPSLKPLSVS YDQATSLRIL NNGHSFNVEF DDSQDKAVIK (crab-eating GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL macaque) VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMSKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK Pan MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY NP_001181853 SEQ. ID. NO. 25 troglodytes DPSLKPLSVS YGQATSLRIL NNGHAFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP HGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK Macaca MSHHWGYGKH NGPEHWHKDF PIAKGQRQSP VDINTHTAKY NP_001182346 SEQ. ID. NO. 26 mulatta DPSLKPLSVS YDQATSLRIL NNGHSFNVEF DDSQDKAVIK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMSKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK Pongo abelii MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002819286 SEQ. ID. NO. 27 DPSLKPLSVC YDQATSLRIL NNGHSFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KCADFTNFDP RGLLPASLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMLKFRKLNF NGEGEPEELM VDNWRPAQPL KKRQIKASFK Callithrix MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002759086 SEQ. ID. NO. 28 jacchus DPSLKPLSVS YDQATSWRIL NNGHSFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGST DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAAQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLESVTWIV LKEPISVSSE QILKFRKLNF SGEGEPEELM VDNWRPAQPL KNRQIKASFK Lemur catta MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDINTGAAKH ADD83028 SEQ. ID. NO. 29 DPSLKPLSVY YEQATSRRIL NNGHSFNVEF DDSQDKAVLK GGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVV DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYLGSLTTP PLLECVTWIV LKEPISVSSE QMMKFRKLSF SGEGEPEELM VDNWRPAQPL KNRQIKASFK Ailuropoda MAHHWGYGKH NGPEHWYKDF PIAKGQRQSP VDIDTKAAIH XP_002916939 SEQ. ID. NO. 30 melanoleuca DPALKALCPT YEQAVSQRVI NNGHSFNVEF DDSQDNAVLK GGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKIG DARPGLQKVL DALDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMLKFRRLNF NKEGEPEELM VDNWRPAQPL HNRQINASFK Eguus MSHHWGYGQH NGPKHWHKDF PIAKGQRQSP VDIDTKAAVH XP_001488540 SEQ. ID. NO. 31 caballus DAALKPLAVH YEQATSRRIV NNGHSFNVEF DDSQDKAVLQ GGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVVGVFLKVG GAKPGLQKVL DVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LREPISVSSE QLLKFRSLNF NAEGKPEDPM VDNWRPAQPL NSRQIRASFK Canis lupus MAHHWGYAKH NGPEHWHKDF PIAKGERQSP VDIDTKAAVH NP_001138642 SEQ. ID. NO. 32 familiaris DPALKSLCPC YDQAVSQRII NNGHSFNVEF DDSQDKTVLK GGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHL VHWNTKYGEF GKAVQQPDGL AVLGIFLKIG GANPGLQKIL DALDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPISVSSE QMLKFRKLNF NKEGEPEELM MDNWRPAQPL HSRQINASFK Oryctolagus MSHHWGYGKH NGPEHWHKDF PIANGERQSP IDIDTNAAKH NP_001182637 SEQ. ID. NO. 33 cuniculus DPSLKPLRVC YEHPISRRII NNGHSFNVEF DDSHDKTVLK EGPLEGTYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHL VHWNTKYGDF GKAVKHPDGL AVLGIFLKIG SATPGLQKVV DTLSSIKTKG KSVDFTDFDP RGLLPESLDY WTYPGSLTTP PLLECVTWIV LKEPITVSSE QMLKFRNLNF NKEAEPEEPM VDNWRPTQPL KGRQVKASFV Ailuropoda GPEHWYKDFP IAKGQRQSPV DIDTKAAIHD PALKALCPTY EFB24165 SEQ. ID. NO. 34 melanoleuca EQAVSQRVIN NGHSFNVEFD DSQDNAVLKG GPLTGTYRLI QFHFHWGSSD GQGSEHTVDK KKYAAELHLV HWNTKYGDFG KAVQQPDGLA VLGIFLKIGD ARPGLQKVLD ALDSIKTKGK SADFTNFDPR GLLPESLDYW TYPGSLTTPP LLECVTWIVL KEPISVSSEQ MLKFRRLNFN KEGEPEELMV DNWRPAQPLH NRQINASFK Sus scrofa MSHHWGYDKH NGPEHWHKDF PIAKGDRQSP VDINTSTAVH XP_001927840.1 SEQ. ID. NO. 35 DPALKPLSLC YEQATSQRIV NNGHSFNVEF DSSQDKGVLE GGPLAGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHL VHWNTKYKDF GEAAQQPDGL AVLGVFLKIG NAQPGLQKIV DVLDSIKTKG KSVEFTGFDP RDLLPGSLDY WTYPGSLTTP PLLESVTWIV LREPISVSSG QMMKFRTLNF NKEGEPEHPM VDNWRPTQPL KNRQIRASFQ Callithrix MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002759087 SEQ. ID. NO. 36 jacchus DPSLKPLSVS YDQATSWRIL NNGHSFNVEF DDSQDKAVLK GGPLDGTYRL IQLHLVHWNT KYGDFGKAAQ QPDGLAVLGI FLKVGSAKPG LQKVVDVLDS IKTKGKSADF TNFDPRGLLP ESLDYWTYPG SLTTPPLLES VTWIVLKEPI SVSSEQILKF RKLNFSGEGE PEELMVDNWR PAQPLKNRQI KASFK Mus MSHHWGYSKH NGPENWHKDF PIANGDRQSP VDIDTATAQH NP_033931 SEQ. ID. NO. 37 musculus DPALQPLLIS YDKAASKSIV NNGHSFNVEF DDSQDNAVLK GGPLSDSYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHL VHWNTKYGDF GKAVQQPDGL AVLGIFLKIG PASQGLQKVL EALHSIKTKG KRAAFANFDP CSLLPGNLDY WTYPGSLTTP PLLECVTWIV LREPITVSSE QMSHFRTLNF NEEGDAEEAM VDNWRPAQPL KNRKIKASFK Bos taurus MSHHWGYGKH NGPEHWHKDF PIANGERQSP VDIDTKAVVQ NP_848667 SEQ. ID. NO. 38 DPALKPLALV YGEATSRRMV NNGHSFNVEY DDSQDKAVLK DGPLTGTYRL VQFHFHWGSS DDQGSEHTVD RKKYAAELHL VHWNTKYGDF GTAAQQPDGL AVVGVFLKVG DANPALQKVL DALDSIKTKG KSTDFPNFDP GSLLPNVLDY WTYPGSLTTP PLLESVTWIV LKEPISVSSQ QMLKFRTLNF NAEGEPELLM LANWRPAQPL KNRQVRGFPK Oryctolagus GKHNGPEHWH KDFPIANGER QSPIDIDTNA AKHDPSLKPL AAA80531 SEQ. ID. NO. 39 cuniculus RVCYEHPISR RIINNGHSFN VEFDDSHDKT VLKEGPLEGT YRLIQFHFHW GSSDGQGSEH TVNKKKYAAE LHLVHWNTKY GDFGKAVKHP DGLAVLGIFL KIGSATPGLQ KVVDTLSSIK TKGKSVDFTD FDPRGLLPES LDYWTYPGSL TTPPLLECVT WIVLKEPITV SSEQMLKFRN LNFNKEAEPE EP Rattus MSHHWGYSKS NGPENWHKEF PIANGDRQSP VDIDTGTAQH NP062164 SEQ. ID. NO. 40 norvegicus DPSLQPLLIC YDKVASKSIV NNGHSFNVEF DDSQDFAVLK EGPLSGSYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHL VHWNTKYGDF GKAVQHPDGL AVLGIFLKIG PASQGLQKIT EALHSIKTKG KRAAFANFDP CSLLPGNLDY WTYPGSLTTP PLLECVTWIV LKEPITVSSE QMSHFRKLNF NSEGRAEELM VDNWRPAQPL KNRKIKASFK

TABLE D3 Exemplary Type VII Carbonic Anhydrases Accession Organism Sequence Number SEQ. ID. NO Human MSLSITNNGH SVQVDFNDSD DRTVVTGGPL EGPYRLKQFH   SEQ. ID. NO. 41 FHWGKKHDVG SEHTVDGKSF PSELHLVHWN AKKYSTFGEA ASAPDGLAVV GVFLETGDEH PSMNRLTDAL YMVRFKGTKA QFSCFNPKCL LPASRHYWTY PGSLTTPPLS ESVTWIVLRE PICISERQMG KFRSLLFTSE DDERIHMVNN FRPPQPLKGR VVKASFRA Pongo MTGHHGWGYG QDDGPSHWHK LYPIAQGDRQ SPINIISSQA XP_002826555 SEQ. ID. NO. 42 abelii VYSPSLQPLE LSYEACMSLS ITNNGHSVQV DFNDSDDRTV VTGGPLEGPY RLKQFHFHWG KKHDVGSEHT VDGKSFPSEL HLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMN RLTDALYMVR FKGTKAQFSCFNPKSLLPAS RHYWTYPGSL TTPPLSESVT WIVLREPICI SERQMGKFRS LLFTSEDDER IHMVNNFRPP QPLKGRVVKA SFRA Pan MEFGLSPELS PSRCFKRLLR GSERGRSRSP NERTEPTGQV XP_001143159.1 SEQ. ID. NO. 43 troglodytes HGCGDGSGMT GHHGWGYGQD DGPSHWHKLY PIAQGDRQSP INIISSQAVY SPSLQPLELS YEACMSLSIT NNGHSVQVDF NDSDDRTVVT GGPLEGPYRL KQFHFHWGKK HDVGSEHTVD GKSFPSELHL VHWNAKKYST FGEAASAPDG LAVVGVFLET GDEHPSMNRL TDALYMVRFK GTKAQFSCFN PKCLLPASRH YWTYPGSLTT PPLSESVTWI VLREPICISE RQMRKFRSLL FTSEDDERIH MVNNFRPPQP LKGRVVKASF RA Callithrix MTGHHGWGYG QDDGPSHWHK LYPIAQGDRQ SPINIISSQA XP_002761099 SEQ. ID. NO. 44 jacchus VYSPSLQPLE LSYEACMSLS ITNNGHSVQV DFNDSDDRTV VTGGPLEGPY RLKQFHFHWG KKHDVGSEHT VDGKSFPSEL HLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMN RLTDALYMVR FKGTKAQFSC FNPKCLLPAS WHYWTYPGSL TTPPLSESVT WIVLREPICI SERQMGKFRS LLFTSEDDER VHMVNNFRPP QPLKGRVVKA SFRA Ailuropoda GPSQWHKLYP IAQGDRQSPI NIVSSQAVYS PSLKPLELSY EFB15849 SEQ. ID.NO. 45 melanoleuca EACISLSIAN NGHSVQVDFN DSDDRTVVTG GPLDGPYRLK QFHFHWGKKH SVGSEHTVDG KSFPSELHLV HWNAKKYSTF GEAASAPDGL AVVGVFLETG DEHPSMNRLT DALYMVRFKG TKAQFSCFNP KCLLPASRHY WTYPGSLTTP PLSESVTWIV LREPISISER QMEKFRSLLF TSEDDERIHM VNNFRPPQPL KGRVVKASFR A Canis MTGHHCWGYG QNDEIQASLS PSLSTPAGPS QWHKLYPIAQ XP_546892 SEQ. ID. NO. 46 familiaris GDRQSPINIV SSQAVYSPSL KPLELSYEAC ISLSITNNGH SVQVDFNDSD DRTAVTGGPL DGPYRLKQLH FHWGKKHSVG SEHTVDGKSF PSELHLVHWN AKKYSTFGEA ASAPDGLAVV GIFLETGDEH PSMNRLTDAL YMVRFKGTKA QFSCFNPKCL LPASRHYWTY PGSLTTPPLS ESVTWIVLRE PISISERQME KFRSLLFTSE EDERIHMVNN FRPPQPLKGR VVKASFRA Bos taurus MTGHHGWGYG QNDGPSHWHK LYPIAQGDRQ SPINIVSSQA XP_002694851 SEQ. ID. NO. 47 VYSPSLKPLE ISYESCTSLS IANNGHSVQV DFNDSDDRTV VSGGPLDGPY RLKQFHFHWG KKHGVGSEHT VDGKSFPSEL HLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMN RLTDALYMVR FKGTKAQFSC FNPKCLLPAS RHYWTYPGSL TTPPLSESVT WIVLREPIRI SERQMEKFRS LLFTSEEDER IHMVNNFRPP QPLKGRVVKA SFRA Rattus MTVLWWPMLR EELMSKLRTG GPSNWHKLYP IAQGDRQSPI EDL87229 SEQ. ID. NO. 48 norvegicus NIISSQAVYS PSLQPLELFY EACMSLSITN NGHSVQVDFN DSDDRTVVAG GPLEGPYRLK QLHFHWGKKR DVGSEHTVDG KSFPSELHLV HWNAKKYSTF GEAAAAPDGL AVVGIFLETG DEHPSMNRLT DALYMVRFKD TKAQFSCFNP KCLLPTSRHY WTYPGSLTTP PLSESVTWIV LREPIRISER QMEKFRSLLF TSEDDERIHM VNNFRPPQPL KGRVVKASFQ S Oryctolagus MTGHHGWGYG QDDGGRPSHW HKLYPIAQGD RQSPINIVSS XP_002711604 SEQ. ID. NO. 49 cuniculus QAVYSPGLQP LELSYEACTS LSIANNGHSV QVDFNDSDDR TVVTGGPLEG PYRLKQFHFH WGKRRDAGSE HTVDGKSFPS ELHLVHWNAR KYSTFGEAAS APDGLAVVGV FLETGNEHPS MNRLTDALYM VRFKGTKAQF SCFNPKCLLP SSRHYWTYPG SLTTPPLSES VTWIVLREPI SISERQMEKF RSLLFTSEDD ERVHMVNNFR PPQPLRGRVV KASFRA Mus GQDDGPSNWH KLYPIAQGDR QSPINIISSQ AVYSPSLQPL AAG16230.1 SEQ. ID. NO. 50 musculus ELFYEACMSL SITNNGHSVQ VDFNDSDDRT VVSGGPLEGP YRLKQLHFHW GKKRDMGSEH TVDGKSFPSE LHLVHWNAKK YSTFGEAAAA PDGLAVVGVF LETGDEHPSM NRLTDALYMV RFKDTKAQFS CFNPKCLLPT SRHYWTYPGS LTTPPLSESV TWIVLREPIR ISERQMEKFR SLLFTSEDDE RIHMVDNFRP PQPLKGRVVK ASFQA Monodelphis MTGHHGWGYG QEDGPSEWHK LYPIAQGDRQ SPIDIVSSQA XP_001364411.1 SEQ. ID. NO. 51 domestic VYDPTLKPLV LAYESCMSLS IANNGHSVMV EFDDVDDRTV VNGGPLDGPY RLKQFHFHWG KKHSLGSEHT VDGKSFSSEL HLVHWNGKKY KTFAEAAAAP DGLAVVGIFL ETGDEHASMN RLTDALYMVR FKGTKAQFNS FNPKCLLPMN LSYWTYPGSL TTPPLSESVT WIVLKEPITI SEKQMEKFRS LLFTAEEDEK VRMVNNFRPP QPLKGRVVQA SFRS Gallus MTGHHSWGYG QDDGPAEWHK SYPIAQGNRQ SPIDIISAKA XP_414152.1 SEQ. ID. NO. 52 gallus VYDPKLMPLV ISYESCTSLN ISNNGHSVMV EFEDIDDKTV ISGGPFESPF RLKQFHFHWG AKHSEGSEHT IDGKPFPCEL HLVHWNAKKY ATFGEAAAAP DGLAVVGVFL EIGKEHANMN RLTDALYMVK FKGTKAQFRS FNPKCLLPLS LDYWTYLGSL TTPPLNESVI WVVLKEPISI SEKQLEKFRM LLFTSEEDQK VQMVNNFRPP QPLKGRTVRA SFKA Taeniopygia MTGQHSWGYG QADGPSEWHK AYPIAQGNRQ SPIDIDSARA XP_002190292.1 SEQ. ID. NO. 53 guttata VYDPSLQPLL ISYESCSSLS ISNTGHSVMV EFEDTDDRTA ISGGPFQNPF RLKQFHFHWG TTHSQGSEHT IDGKPFPCEL HLVHWNARKY TTFGEAAAAP DGLAVVGVFL EIGKEHASMN RLTDALYMVK FKGTKAQFRG FNPKCLLPLS LDYWTYLGSL TTPPLNESVT WIVLKEPIRI SVKQLEKFRM LLFTGEEDQR IQMANNFRPP QPLKGRIVRA SFKA

TABLE D4 Exemplary Type XIII Carbonic Anhydrases Accession Organism Sequence Number SEQ. ID. NO Human MSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTKEVK NP_940986.1 SEQ. ID. NO. 54 YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTENKSVL RGGPLTGSYR LRQVHLHWGS ADDHGSEHIV DGVSYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQK ITDTLDSIKE KGKQTRFTNF DLLSLLPPSW DYWTYPGSLT VPPLLESVTW IVLKQPINIS SQQLAKFRSL LCTAEGEAAA FLVSNHRPPQ PLKGRKVRAS FH Pan MSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTKEVK XP_001169377.1 SEQ. ID. NO. 55 troglodytes YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTENKSVL RGGPLTGSYR LRQFHLHWGS ADDHGSEHIV DGVSYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQK ITDTLDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVTW IVLKQPINIS SQQLAKFRSL LCTAEGEAAA FLVSNHRPPQ PLKGRKVRAS FH Macaca MSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTQEVK XP_001095487.1 SEQ. ID. NO. 56 mulatta YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVL RGGPLAGSYR LRQFHLHWGS ADDHGSEHIV DGVSYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQK ITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVIW IVLKQPINVS SQQLAKFRSL LCTAEGEAAA FLLSNHRPPQ PLKGRKVRAS FR Oryctolagus MSRISWGYGE HNGPIHWNQF FPIADGDQQS PIEIKTKEVK XP_002710714.1 SEQ. ID. NO. 57 cuniculus YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVL RGGPLTGNYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEYNSQLQK ITDILDSIKE KGKQTRFTNF DPLSLLPSSW DYWTYPGSLT VPPLLESVTW IVLKQPINIS SQQLAKFRSL LCSAEGESAA FLLSNHRPPQ PLKGRKVRAS FH Ailuropoda MSRLSWGYGE HNGPIHWNKF FPIADGDQQS PIEIKTKEVK XP_002916937.1 SEQ. ID. NO. 58 melanoleuca YDSSLRPLSI KYDANSAKII SNSGHSFSVD FDDTEDKSVL RGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEHNSQLQK ITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVTW IVLKQPINIS SEQLATFRTL LCTAEGEAAA FLLSNHRPPQ PLKGRKVRAS FH Sus scrofa MSRFSWGYGE HNGPVHWNEF FPIADGDQQS PIEIKTKEVK XP_001924497.1 SEQ. ID. NO. 59 YDSSLRPLSI KYDPSSAKII SNSGHSFSVD FDDTEDKSVL RGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVKYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEHNSQLQK ITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVTW IILKQPINIS SQQLATFRTL LCTKEGEEAA FLLSNHRPLQ PLKGRKVRAS FH Callithrix MSRLSWGYGE HNGPIHWNEF FPIADGDRQS PIEIKAKEVK XP_002759085.1 SEQ. ID. NO. 60 jacchus YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVL HGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH VVHWNSEKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQK IIDILDSIKE KGKOIRFTNF DPLSLFPPSW DYWTYSGSLT VPPLLESVTW ILLKQPINIS SQQLAKFRSL LCTAEGEAAA FLLSNYRPPQ PLKGRKVRAS FR Rattus MARLSWGYDE HNGPIHWNEL FPIADGDQQS PIEIKTKEVK NP_001128465.1 SEQ. ID. NO. 61 norvegicus YDSSLRPLSI KYDPASAKII SNSGHSFNVD FDDTEDKSVL RGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH VVHWNSDKYP SFVEAAHESD GLAVLGVFLQ IGEHNPQLQK ITDILDSIKE KGKQTRFTNF DPLCLLPSSW DYWTYPGSLT VPPLLESVTW IVLKQPISIS SQQLARFRSL LCTAEGESAA FLLSNHRPPQ PLKGRRVRAS FY Mus MARLSWGYGE HNGPIHWNEL FPIADGDQQS PIEIKTKEVK NP_078771.1 SEQ. ID. NO. 62 musculus YDSSLRPLSI KYDPASAKIISNSGHSFNVD FDDTEDKSVL RGGPLTGNYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH VVHWNSDKYP SFVEAAHESD GLAVLGVFLQ IGEHNPQLQK ITDILDSIKE KGKQTRFTNFDPLCLLPSSW DYWTYPGSLT VPPLLESVTW IVLKQPISIS SQQLARFRSL LCTAEGESAA FLLSNHRPPQ PLKGRRVRAS FY Canis MPPRRHGPNT FLSAGTKGQQ NFWTKNQKSG PIHWNKFFPI XP_544159 SEQ. ID. NO. 63 familiaris ADGDQQSPIE IKTKEVKYDS SLRPLSIKYD ANSAKIISNS GHSFSVDFDD TEDKSVLRGG PLTGSYRLRQ FHLHWGSADD HGSEHVVDGV RYAAELHVVH WNSDKYPSFV EAAHEPDGLA VLGVFLQIGE HNSQLQKITD ILDSIKEKGK QTRFTNFDPL SLLPPSWDYW TYPGSLTVPP LLESVTWIVL KQPINISSQQ LATFRTLLCT AEGEAAAFLL SNHRPPQPLK GRKVRASFH Eguus MSGPVHWNEF FPIADGDQQS PIEIKTKEVK YDSSLRPLTI XP_001489984.2 SEQ. ID. NO. 64 caballus KYDPSSAKII SNSGHSFSVG FDDTENKSVL RGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELH IVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ VGEHNSQLQK ITDTLDSIKE KGKQTLFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVTW IILKQPINIS SQQLVKFRTL LCTAEGETAA FLLSNHRPPQ PLKGRKVRAS FR Bos taurus MSGFSWGYGE RDGPVHWNEF FPIADGDQQS PIEIKTKEVR XP_002692875.1  SEQ. ID. NO. 65 YDSSLRPLGI KYDASSAKII SNSGHSFNVD FDDTDDKSVL RGGPLTGSYR LRQFHLHWGS TDDHGSEHVV DGVRYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGIFLQ IGEHNPQLQK ITDILDSIKE KGKQTRFTNF DPVCLLPPCR DYWTYPGSLT VPPLLESVTW IILKQPINIS SQQLAAFRTL LCSREGETAA FLLSNHRPPQ PLKGRKVRAS FR Monodelphis MSRLSWGYCE HNGPVHWSEL FPIADGDYQS PIEINTKEVK XP_001366749.1 SEQ. ID. NO. 66 domestica YDSSLRPLSI KYDPASAKII SNSGHSFSVD FDDSEDKSVL RGGPLIGTYR LRQFHLHWGS TDDQGSEHTV DGMKYAAELH VVHWNSDKYP SFVEAAHEPD GLAVLGIFLQ TGEHNLQMQK ITDILDSIKE KGKQIRFTNF DPATLLPQSW DYWTYPGSLT VPPLLESVTW IVLKQPITIS SQQLAKFRSL LYTGEGEAAA FLLSNYRPPQ PLKGRKVRAS FR Ornithorhynchus MKKGVGSFYE LAVNRWSVVN RVQIMIVESI TEPLLCGSRA XP_001507177.1 SEQ. ID. NO. 67 anatinus LALTLSPTQA LAVAPALALA VVQALALTVV QALALAVSPA LALSVAPALA LAVVQALALA VVQALALAVA QALALAVAQA LALAVAQALA LALPQALALT LPQALALTLS PTLALSVAPA LALAVAPALA LADSPALALA LARPHPSSGS SPALDCELVL FGDCHTVLLK WMRMGNYSSV SPLEERNSSC PLGPIHWNEL FPIADGDRQS PIEIKTKEVK YDSSLRPLSI KYDPTSAKII SNSGHSFSVD FDDTEDKSVL RGGPLSGTYR LRQFHFHWGS ADDHGSEHTV DGMEYSAELH VVHWNSDKYS SFVEAAHEPD GLAVLGIFLK RGEHNLQLQK ITDILDAIKE KGKQMRFTNF DPLSLLPLTR DYWTYPGSLT VPPLLESVIW IIFKQPISIS SQQLAKFRNL LYTAEGEAAD FMLSNHRPPQ PLKGRKVRAS FRS

TABLE D5 Exemplary CA II DNA expression constructs for chloroplast expression ATGTCCCATC ACTGGGGGTA CGGCAAACAC AACGGACCTG AGCACTGGCA TAAGGACTTC SEQ. ID. NO. 94 CCCATTGCCA AGGGAGAGCG CCAGTCCCCT GTTGACATCG ACACTCATAC AGCCAAGTAT (human cDNA GACCCTTCCC TGAAGCCCCT GTCTGTTTCC TATGATCAAG CAACTTCCCT GAGGATCCTC sequence) AACAATGGTC ATGCTTTCAA CGTGGAGTTT GATGACTCTC AGGACAAAGC AGTGCTCAAG GGAGGACCCC TGGATGGCAC TTACAGATTG ATTCAGTTTC ACTTTCACTG GGGTTCACTT GATGGACAAG GTTCAGAGCA TACTGTGGAT AAAAAGAAAT ATGCTGCAGA ACTTCACTTG GTTCACTGGA ACACCAAATA TGGGGATTTT GGGAAAGCTG TGCAGCAACC TGATGGACTG GCCGTTCTAG GTATTTTTTT GAAGGTTGGC AGCGCTAAAC CGGGCCTTCA GAAAGTTGTT GATGTGCTGG ATTCCATTAA AACAAAGGGC AAGAGTGCTG ACTTCACTAA CTTCGATCCT CGTGGCCTCC TTCCTGAATC CTTGGATTAC TGGACCTACC CAGGCTCACT GACCACCCCT CCTCTTCTGG AATGTGTGAC CTGGATTGTG CTCAAGGAAC CCATCAGCGT CAGCAGCGAG CAGGTGTTGA AATTCCGTAA ACTTAACTTC AATGGGGAGG GTGAACCCGA AGAACTGATG GTGGACAACT GGCGCCCAGC TCAGCCACTG AAGAACAGGC AAATCAAAGC TTCCTTCAAA TAA gaattcATGTCtCATCAtTGGGGtTAtGGtAAACACAAtGGtCCTGAaCACTGGCATAAaGACTT SEQ. ID. NO. 108 tCCaATTGCaAAaGGtGAaCGtCAaTCaCCTGTTGAtATtGACACTCATACAGCtAAaTATGACC (Optimized for CTTCttTaAAaCCatTaTCTGTTTCaTATGATCAAGCAACTTCttTacGtATttTaAACAATGGT chloroplast CATGCTTTtAAtGTaGAaTTTGATGACTCTCAaGAtAAAGCAGTatTaAAaGGtGGtCCatTaGA Expression) TGGtACTTACcGtTTaATTCAaTTTCACTTTCACTGGGGTTCAtTaGATGGtCAAGGTTCAGAaC ATACTGTaGATAAAAAaAAATATGCTGCAGAAtTaCACTTaGTTCACTGGAACACaAAATATGGt GATTTTGGtAAAGCTGTaCAaCAACCTGATGGttTaGCtGTTtTAGGTATTTTTTTaAAaGTTGG tAGtGCTAAACCaGGtCTTCAaAAAGTTGTTGATGTatTaGATTCaATTAAAACAAAaGGtAAaA GTGCTGACTTtACTAAtTTCGATCCTCGTGGttTaCTTCCTGAATCtTTaGATTACTGGACaTAt CCAGGtTCAtTaACaACaCCTCCTCTTtTaGAATGTGTaACaTGGATTGTatTaAAaGAACCaAT tAGtGTaAGtAGtGAaCAaGTaTTaAAATTCCGTAAACTTAAtTTCAATGGtGAaGGTGAACCaG AAGAAtTaATGGTtGAtAACTGGCGtCCAGCTCAaCCAtTaAAaAAtcGtCAAATtAAAGCTTCa TTCAAATAAgcatgc

TABLE D6 Codons in Human CA II optimized for expression in chloroplast of Chlamydomonas reinhardtii Number of codons No. of amino Expected Amino Total that were acids of ratio acid number optimized each codon of codons Ser(S) 18 12 TCT TCA 1:1:1 AGT (7:7:5) Phe(F) 12 3 TIT TTC (8:4) 2:1 Leu(L) 26 19 TIA CTT (21:5) 5:1 Val(V) 17 10 GTT GTA (8:9) 1:1 Pro(P) 17 6 CCT CCA (8:9) 3:4 Thr(T) 12 5 ACT ACA (5:7) 2:3 Ala(A) 13 3 GCT GCA (9:4) 2:1 Tyr(Y) 8 2 TAT TAC (6:2) 2:1 His(H) 12 1 CAT CAC (6:6) 1:1 Asn(N) 10 4 AAT AAC (7:3) 2.5 1 A(D) 19 3 GAT GAC (14:5) 2.5 1 Ile(I) 9 4 ATT (9) 1 Met(M) 2 0 ATG (2) 1 Gln(Q) 11 7 CAA (11) 1 Glu(E) 13 6 GAA (13) 1 Lys(K) 24 11 AAA (24) 1 Cys(C) 1 0 TGT (1) 1 Tf£_(W) 7 0 TGG (7) 1 Gly(G) 22 17 GGT (22) 1 Arg(R) 7 5 CGT (7) 1

TABLE D7 Exemplary algal bicarbonate transporter types Transport Substrate Photosynthetic Type Mechanism affinity Flux rate affinity ko.6 BicA Na+ Low- High 90-170 μM dependent medium HC0³⁻ SbtA Na+ High Low <5 μM HC0₃ dependent HC0³⁻ uptake BicA Na+ Low- High 90-170 μM dependent medium HC0³⁻ SbtA Na+ High Low <5 μM HC0₃ dependent HC0³⁻ uptake

TABLE D8 Exemplary plasma membrane localized Bicarbonate transporters Accession Organism Sequence Number SEQ. ID. NO Chlamydomonas MLPGLGVILL VLPMQYYFGY KIVQIKLQNA KHVALRSAIM EDP07736.1 SEQ. ID. NO. 77 reinhardtii QEVLPAIKLV KYYAWEQFFE NQISKVRREE IRLNFWNCVM KVINVACVFC VPPMTAFVIF TTYEFQRARL VSSVAFTTLS LFNILRFPLV VLPKALRAVS EANASLQRLE AYLLEEVPSG TAAVKTPKNA PPGAVIENGV FHHPSNPNWH LHVPKFEVKP GQVVAVVGRI AAGKSSLVQA ILGNMVKEHG SFNVGGRISY VPQNPWLQNL SLRDNVLFGE QFDENKYTDV IESCALTLDL QILSNGDQSK AGIRGVNFSG GQRQRVNLAR CAYADADLVL LDNALSAVDH HTAHHIFDKC IKGLFSDKAV VLVTHQIEFM PRCDNVAIMD EGRCLYFGKW NEEAQHLLGK LLPITHLLHA AGSQEAPPAP KKKAEDKAGP QKSQSLQLTL APTSIGKPTE KPKDVQKLTA YQAALIYTWY GNLFLVGVCF FFFLAAQCSR QISDFWVRWW VNDEYKKFPV KGEQDSAATT FYCLIYLLLV GLFYIFMIFR GATFLWWVLK SSETIRRKAL HNVLNAPMGF FLVTPVGDLL LNFTKDQDIM DENLPDAVHF MGIYGLILLA TTITVSVTIN FFAAFTGALI IMTLIMLSIY LPAATALKKA RAVSGGMLVG LVAEVLEGLG VVQAFNKQEY FIEEAARRTN ITNSAVFNAE ALNLWLAFWC DFIGACLVGV VSAFAVGMAK DLGGATVGLA FSNIIQMLVF YTWVVRFISE SISLFNSVEG MAYLADYVPH DGVFYDQRQK DGVAKQIVLP DGNIVPAASK VQVVVDDAAL ARWPATGNIR FEDVWMQYRL DAPWALKGVT FKINDGEKVG AVGRTGSGKS TTLLALYRMF ELGKGRILVD GVDIATLSLK RLRTGLSIIP QEPVMFTGTV RSNLDPFGEF KDDAILWEVL KKVGLEDQAQ HAGGLDGQVD GTGGKAWSLG QMQLVCLARA ALRAVPILCL DEATAAMDPH TEAIVQQTIK KVFDDRTTIT IAHRLDTIIE SLMEYESPSK LLANRDSMFS KLVDKTGPAA AAALRKMAED FWSTRSAQGR NQ Volvox carteri MGTISHPARG NDPTAGFFNK EAFGWMFKHV SEARKNGDID XP_002950646.1 SEQ. ID. NO. 69 f. nagariensis LDKMGMPPEN HAHEAYDMFA SNWAAEMKLK DSGAKPSLVR ALRKSFGLVY LLGGVFKCFW STFVITGAFY FVRSLLAHVN GIKDGRLYSK TVSGWCLMAG FTLDAWLLGL SLQRMGYICM SVGIRARAAL VQAVTHKAFR LSSVRADQSA AIVNFVSSDI QKIYDGALEF HYLWTAPFEA AAILALLGYL TNDSMLPGLG VILLVLPLQY FFGYKIIQIK LQNAKHVALR SSILQEVLPA IKLVKYYAWE QFFEDEISKI RREEMRLSFW NAMMKVINVA CVFCVPPMTA FVIFTTYEFQ KARLVSGVAF TTLSLFNILR FPLVVLPKAL RAVSEAHASL QRLESYLLED VPQGTASGGK SSKSSAPGVH IDNAVYHHPS NPNWHLHVPR FDVRPGQVVA VVGRIGAGKS SLVQAILGNM VKEHGSQQVG GRISYVPQNP WLQNLSIRDN VTFGEGWDEN KYEAVIDACA LTMDLQILPQ GDQSKAGIRG VNFSGGQRQR VNLARCAYAD ADLVLLDNAL SAVDHHTAHH IFDKCIKGLF SDKAVVLITH QIEFMPRCDA VAIMDEGRCL YFGKWNEESQ HLLGKLLPIT HLLHAAGSQE APPAAPKKKD DKATPQKSQS LQLTLAPTSI GKPTQKDTKA APKLTAFKAA LIYTYYGNIL LVFVCFITFL AAQTCRQMSD FWVRWWVNDE YKHFPKRTGV REESATKFYA LIYLLLVGLF YFTMVARGST FLWWVLRSSE NIRKKALNNV LNAPMGFFLV TPVGDLLLNF TKDQDIMDEN LPDAIHFMGI YGLILLATTI TVSVTINFFG AFTGFLIIMT LIMLAIYLPA ATALKKARAV SGGQLVGLVA EVLEGLNVVQ AFSKQEYFIE EAARRTDVTN AAVFNAESLN LWLAFWCDLI GASLVGVVSA FAVGLKDQLG AATVGLAFSN IIQMLVFYTW VVRFIAESIS LFNSVEAMAW LADYVPKDGI FYDQKQLDGV AKSITLPDGQ IVPATSKVQV VVDDAALARW PATGNIRFED VWMQYRLDAA WALKGVTFKI NDGEKVGAVG RTGSGKSTTL LALYRMFELG KGRILIDGVD IATLSLKRLR TGLSIIPQEP VMFTGTVRSN LDPFGEFKDD SVLWEVLQKV GLEAQAQHAG GLDGRVDGTG GKAWSLGQMQ LVCLARAALR AVPILCLDEA TAAMDPHTEQ VVQETIKKVF DDRTTITIAH RLDTIIESDK VLVMEAGELK EFAPPAQLLA NRETMFSKLV DKTGPAAAAA LRKMADEHFS KSQARAAAQR H Chlorella MVPLLAQRGR IRSQAPRTWH PDPQPLHAER SRQCPGRGVR EFN52914.1 SEQ. ID. NO. 70 variabilis AAAKRGGGSG GATHKSKKSK ELDEVAAFEQ LMCDWDDAFA ADCYDNERAA RMARLAEEGY QHHGRGFVFV RSRLDKRSRK ARNDSGASKG FGAAAKALSV EQGTPLENNP QLHLLSWTAC YIASSQLDSL GGLFSTQEGV LLPDSGSLLT DGGSGASGSN AADAVGELQR VLRGQDLSQL RGYVGAPPQA RPASGSDDDG SSTTGSNNGA AGEGSEVEEG TAMGGIRRYE PESGELVVLL SCKIGGKPAV GAELLAVAQA EDGKHAPGAS PDTRLCKEPS QSAFDLWSFG WMNKIVPAAR RGEVEVADLP LPEAQQAEPC YEELNTNWEA AVQEAKKAGK EPKLMKVLWK TYGKDIVLAG IFKLMWSVFV ILGAYYFTRS ILMCIRTLEG KDDSIYDTEW KGWVLTGFFF LDAWLLGMML QRMAFNCLKV GIKARAALTT MIARKCYNMA HLTKDTAAEA VGFVASDINK VFEGIQEVHY LWGAPVEAGA ILALLGTLVG VYCIGGVIIV CMVVPLQYYF GYKIIKNKIK NAPNVTERWS IIQEILPAMK LVKYYAWERF FEKHVADMRT RERHYMFWNA VVKTVNVTMV FGVPPMVTFA VLVPYELWHV DSSTSEPYIK PQTAFTMLSL FNVLRFPLVV LPKAMRCVSE ALRSVGNLEK FLAEPVAPRQ DLEGKPGAQL SKAVLRHEMD TSGFTLRVPE FSVKAGELVA VVGRVGAGKS SILQAMLGNM QTASGLAKCQ HSASSCLPFL VEGTAHSGGR IAYVPQTAWC QNLSLRDNIT FGQPWDEAKY KQVIHACALE LDLAILAAGD QSKAGLRGIN LSGGQRQRLN LARCAYFDGD LVLLDNALSA VDHHTAHHIF EHCVRGMFRD KATVLVTHQV EFLPQCDKVA IMDDGTCVYF GPWNAAAQQL LSKYLPASHL LAAGGNAEQP RDTKKKVVKK EETKKTEDAG KAKRVHSASL TLKSALWEYC WDARWIIFCL SLFFFLTAQA SRQLADYFIR WWTRDHYNKY GVLCIDEGDN PCGPLFYVQY YGILGLLCFI VLMAFRGAFL YTWSLGASYR QHEKSIHRVL YAPLGFFLTT PVGDLLVSFT KDQDVMDDAL PDALYYAGIY GLILLATAIT VSVTIPLFSA LAGGLFVVSG IMLAIYLPAA THLKKLRMGT SGDVVTLIAE ALDGLGVIQA YGKQAYFTTI TSQYVNDAHR ALFGAESLNL WLAFICDFFG ACMVLSVACF GIGQWSTLGS SSVGLAFSQS IQMLVFYTWS IRLVAECIGL FGSAEKIAWL ANHTPQEAGS LDPPSLPGSG ETKAAPKKRG TAGKFLPPLK DEDLAIVPTG GPKLPSGWPR TGVLEFNQVV MKYAPHLPPA LRGVSFKVKS GDKVGVVGRT GSGKSTLLLA LYRMFNLESG AITLDGIDIS TLTLEQLRRG LSVIPQEPTV FSGTVRTNLD PFGEFGADAI LWEALRDCGL EEQVKACGGL DAKLDGTGGN AWSIGQQQLM CLARAALKKV PVLCLDEATA AMDPHTEAHV LEIIERIFSD RTMLTIAHRL DNVIRSDLVV VMDAGQVCEM GTPDELLANP_QSAFSQLVDK TGAASAAALR KMAADFLDER ARGQKLGFKP RPSLEESHIC VAPSPSLILS TLLFPPAFMA NVTALLLPKP VLSHAPVSSQ TVNTYIRLNI IQLQCNVLHP ATKEATWSSR RITFTAHLSS SGSKPPPPLP PLTELPEGRG LDWSSAGYRD GREAIPSPSA KYSAADYGAA GDGVTDDTQA LQVAVAAAHE DDEGGVVYLG AGTFVLTQPL SIAGSNVVIR GAGEDATTIF VPLPLSDVFP GTWSMDASGK VTSPWITRGG FLAFSGRRTK SSDSSTLLAT VAGSVEQGAS VIPVDSTAEF RLGQWVRIII NDASTDASAG GGTLERGSSE VQESETMIAE GATGGGAGVR AQWTGVLHAF EPTVQCSGVE QLTIRFNHSM MAAHLAERGY NAIELEDVVD CWIRQVTILN ADNAIRLRGT DHSTLSGQAC SGGGVVAVVP VWCRRGLPSP ADVTVGVTEL RWEPDTREVN GHHAITVSKG HANLVTRFRI TAPFYHDISL EGGALLNVIS SGGGANLNLD LHRSGPWGNL FSQLGMGLAA RPFDAGGRDG RGAHAGRQNT FWNLQPGDVA AAAPALQPSA AAGDARRLLV DGDSLLHAGT GQARLLRQLE ADDSAEPLLL PSCEFGPLLN FVGGFAGELC KSSGWLVAGL PDDRPDLHAS QVTARLQHGA ADNKTHA Synechococcus MDFLSNFLMD FVKQLQSPTL SFLIGGMVIA ACGSQLQIPE ABB57505.1 SEQ. ID. No. 71 elongatus SICKIIVFML LTKIGLTGGM AIRNSNLTEM VLPALFSVAI PCC 7942.J GILIVFIARY TLARMPKVKT VDAIATGGLF GAVSGSTMAA ALTLLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLN KKKRKEAAFA SAQGAYSKQP VAAGDYSSSS DYPSSRREYA QQESGDHRVK IWPIVEESLQ GPALSAMLLG VALGLFARPE SVYEGFYDPL FRGLLSILML VMGMEAWSRI SELRKVAQWY VVYSIVAPLA HGFIAFGLGM IAHYATGFSM GGVVVLAVIA ASSSDISGPP TLRAGIPSAN PSAYIGASTA IGTPVAIGIA IPLFLGLAQT IGG Synechocystis MDFLSNFLTD FVGQLQSPTL AFLIGGMVIA ALGTQLVIPE NP_441340 SEQ. ID. No.72 sp. AISTIIVFML LTKIGLTGGM AIRNSNLTEM LLPVAFSVIL PCC 6803 GILIVFIARF TLAKLPNVRT VDALATGGLF GAVSGSTMAA ALTTLEESKI SYEAWAGALY PFMDIPALVT AIVVANIYLN KRKRKSAAAS IEESFSKQPV AAGDYGDQTD YPRTRQEYLS QQEPEDNRVK IWPIIEESLQ GPALSAMLLG LALGIFTKPE SVYEGFYDPL FRGLLSILML IMGMEAWSRI GELRKVAQWY VVYSLIAPIV HGFIAFGLGM IAHYATGFSL GGVVVLAVIA ASSSDISGPP TLRAGIPSAN PSAYIGSSTA IGTPIAIGVC IPLFIGLAQT LGAG Nostoc sp. MDFFSLFLMD FVKQLQSPTL GFLIGGMVIA ALGSELIIPE NP_486174 SEQ. ID. No. 73 PCC 712 AICQIIVFML LTKIGLTGGI AIRNSNLTEM VLPAASAVAV GVLVVFIARY TLAKLPKVNT VDAIATGGLF GAVSGSTMAA ALTLLEEQKI QYEAWAAALY PFMDIPALVT AIVVANIYLN KKKRSAAGEY LSKQSVAAGE YPDQQDYPSS RQEYLRKQQS ADNRVKIWPI VKESLQGPAL SAMLLGIALG LFTQPESVYK SFYDPLFRGL LSILMLVMGM EAWSRIGELR KVAQWYVVYS VVAPLVHGFI AFGLGMIAHY ATGFSLGGVV ILAVIAASSS DISGPPTLRA GIPSANPSAY IGASTAIGTP IAIGLAIPLF LGLAQAIGGR Cyanothece sp. MDFWSYFLMD FVKQLQSPTL GFLIGGMVIA ALGSQLVIPE YP_002485721 SEQ. ID. No. 74 PCC 7425 AICQIIVFML LTKIGLTGGM AIRNSNLTEM VLPAAFSVIS GILIVFIARY TLAKLPKVRT VDAIATGGLF GAVSGSTMAA ALTLLEEEKI PYEAWAGALY PFMDIPALVT AIVIANIYLN KKKRRAESEA LSKQEYLGKQ SIVAGDYPAQ QDYPSTRQEY LSKQQGPENN RVKIWPIVQE SLQGPALSAM LLGVALGILT KPESVYESFY DPLFRGLLSI LMLVMGMEAW SRIGELRKVA QWYVVYSVVA PFVHGLIAFG LGMFAHYTMG FSMGGVVVLA VIASSSSDIS GPPTLRAGIP SANPSAYIGA STAIGTPIAI GLCIPFFIGL AQTLGGG Microcysti MDFFSLFVMD FIQQLQSPTL AFLIGGMIIA ALGSELVIPE YP_001661223 SEQ. ID. No. 75 aeruginosa SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMIFAVIV NIES-843 GIIVVFVARY TLANLPKVKV VDAIATGGLF GAVSGSTMAA GLTVLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLN KKKQKEAAYD QESFSKQPVA AGNYSDQQDY PSSRQEYLSQ QQPADNRVKI WPIIEESLRG PALSAMLLGL ALGIFTQPES VYKSFYDPLF RGLLSVLMLV MGMEAWSRVG ELRKVAQWYV VYSVIAPFVH GLIAFGLGMI AHYATGFSWG GVVMLAVIAS SSSDISGPPT LRAGIPSANP SAYIGASTAI GTPVAIGLCI PFFVGLAQAL SGG Anabaena MDFVSLFVKD FIAQLQSPTL AFLIGGMIIA ALGSELVIPE YP_323532 SEQ. ID. No. 86 variabills SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMIFAVIT ATCC 29413 GITIVFISRY TLAKLPKVKV VDAIATGGLF GAVSGSTMAA GLTVLEEQKM AYEAWAGALY PFMDIPALVT AIVIANIYLN KKKRKEAVYS TEQPVAAGDY PDQKDYPSSR QEYLSQQKGD EDNRVKIWPI IEESLRGPAL SAMLLGLALG LFTQPESVYK SFYDPAFRGL LSILMLVMGM EAWSRIGELR KVAQWYVVYS VVAPFVHGLI AFGLGMIAHY TMNFSMGGVV ILAVIASSSS DISGPPTLRA GIPSANPSAY IGASTAVGTP VAIGLCIPFF LGLAQAIGG Cyanothece sp. MDFLSLFVKD FIIQLQSPTL AFLIGGMVIA ALGSELVIPE YP_002371470.1 SEQ. ID. No. 87 PCC 880 SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMICAVIV   GIVVVFIARY TLAKLPKVNV VDAIATGGLF GAVSGSTMAA GLTVLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLN KKKRKATVMQ ESLSKQPVAA GDYPSSRQEY VSQQQPEDNR VKIWPIIEES LRGPALSAML LGLALGILTQ PESVYKGFYD PPFRGLLSIL MLVMGMEAWS RIGELRKVAQ WYVVYSVAAP FIHGLLAFGL GMIAHYTMGF SMGGVVILAV IASSSSDISG PPTLRAGIPS ANPSAYIGAS TAIGTPVAIG LCIPFFVGLA QAIGGF Arthrospia MDFLSGFLTR FLAQLQSPTL GFLIGGMVIA AVNSQLQIPD ZP.06383808.1 SEQ. ID. No. 88  platensis AIYKFVVFML LMKVGLSGGI AIRGSNLTEM LLPAVFALVT str. GIVIVFIGRY TLAKLPNVKT VDAIATAGLF GAVSGSTMAA Paraca ALTLLEEQGM EYEAWAAALY PFMDIPALVS AIVLASIYVS KQKHSDMADE SLSKHESLSK QPVAAGDYPS KPEYPTTRQE YLSQQRGSAN QGVEIWPIIK ESLQGSALSA LLLGLALGLL TRPESVFQSF YEPLFRGLLS ILMLVMGMEA TARLGELRKV AQWYAVYAFI APLLHGLIAF GLGMIAHVVT GFSLGGVVIL AVIASSSSDI SGPPTLRAGI PSANPSAYIG SSTAVGTPVA IALGIPLYIG LAQALMGG

TABLE D9 Exemplary chloroplast envelope localized Bicarbonate transporters Accession Organism Sequence Number SEQ. ID. NO MQTTMTRPCL AQPVLRSRVL RSPMRVVAAS APTAVTTVVT BAD16681.1 SEQ. ID. NO.89 Chlamydomonas SNGNGNGHFQ AATTPVPPTP APVAVSAPVR AVSVLTPPQV reinhardtii YENAINVGAY KAGLTPLATF VQGIQAGAYI AFGAFLAISV GGNIPGVAAA NPGLAKLLFA LVFPVGLSMV TNCGAELFTG NTMMLTCALI EKKATWGQLL KNWSVSYFGN FVGSIAMVAA VVATGCLTTN TLPVQMATLK ANLGFTEVLS RSILCNWLVC CAVWSASAAT SLPGRILALW PCITAFVAIG LEHSVANMFV IPLGMMLGAE VTWSQFFFNN LIPVTLGNTI AGVLMMAIAY SISFGSLGKS AKPATA Volvox carteri MQTTMSVTRP CVGLRPLPVR NVRSLIRAQA APQQVSTAVS XP_002951507.1 SEQ. ID. NO. 79 f. nagariensis TNGNGNGVAA ASLSVPAPVA APAQAVSTPV RAVSVLTPPQ VYENAANVGA YKASLGVLAT FVQGIQAGAY IAFGAFLACS VGGNIPGITA SNPGLAKLLF ALVFPVGLSM VTNCGAELYT GNTMMLTCAI FEKKATWAQL VKNWVVSYAG NFVGSIAMVA AVVATGLMAS NQLPVNMATA KSSLGFTEVL SRSILCNWLV CCAVWSASAA TSLPGRILGL WPPITAFVAI GLEHSVANMF VIPLGMMLGA DVTWSQFFFN NLVPVTLGNT IAGVVMMAVA YSVSYGSLGK TPKPATA

TABLE D10 Transit Peptides Organism SEQ ID NO Name Arabidopsis 8 Rbcs-1a transit thaliana peptide Arabidopsis 14 PGR5 transit thaliana peptide Arabidopsis 15 psaD transit thaliana peptide Arabidopsis 22 DNAJ transit thaliana peptide Cyanophora 102 psaD trasit paradoxa peptide Arabidopsis 104 CAB transit thaliana peptide Arabidopsis 105 PGR5 transit thaliana peptide

TABLE D11 Cyclic Electron Transfer modulator proteins SEQ ID Accession Organism NO Name No. Function Arabidopsis 93 Ferredoxin1 AEE28669.1 cyclic electron thaliana (FD1) transfer modulator protein Arabidopsis 95 Ferredoxin2 AAG40057.1 cyclic electron thaliana (FD2) transfer modulator protein Arabidopsis 96 ferredoxin- AT5G66190 cyclic electron thaliana NADP(+) partial transfer oxidoreductase modulator (FNR1) protein Arabidopsis 97 ferredoxin- BAH19611.1 cyclic electron thaliana NADP(+) transfer oxidoreductase modulator (FNR2) protein

An exemplary optimized DNA sequence for the plasma membrane localized bicarbonate transporter is shown in SEQ ID NO. 91

(SEQ ID NO: 91) atgctgcccg gcctgggcgt catcctgctg gtgctgccca tgcagtacta cttcggctac 60 aagatcgtgc agatcaagct gcagaacgcc aagcacgtcg ccctgcgctc cgccatcatg 120 caggaggtgc tgcccgccat caagctggtc aagtactacg cctgggagca gttctttgag 180 aaccagatca gcaaggtccg ccgcgaggag atccgcctca acttctggaa ctgcgtgatg 240 aaggtcatca acgtggcctg cgtgttctgc gtgccgccca tgaccgcctt cgtcatcttc 300 accacctacg agttccagcg cgcccgcctg gtgtccagcg tcgccttcac caccctgtcg 360 ctgttcaaca ttctgcgctt ccccctggtc gtgctgccca aggccctgcg tgccgtgtcc 420 gaggccaacg cgtctctcca gcgcctggag gcctacctgc tggaggaggt gccctcgggc 480 actgccgccg tcaagacccc caagaacgct ccccccggcg ccgtcatcga gaacggtgtg 540 ttccaccacc cctccaaccc caactggcac ctgcacgtgc ccaagttcga ggtcaagccc 600 ggccaggtcg ttgctgtggt gggccgcatc gccgccggca agtcgtccct ggtgcaggcc 660 atcctcggca acatggtcaa ggagcacggc agcttcaacg tgggcggccg catctcctac 720 gtgccgcaga acccctggct gcagaacctg tccctgcgtg acaacgtgct gtttggcgag 780 cagttcgatg agaacaagta caccgacgtc atcgagtcct gcgccctgac cctggacctg 840 cagatcctgt ccaacggtga ccagtccaag gccggcatcc gcggtgtcaa cttctccggt 900 ggccagcgcc agcgcgtgaa cctggcccgc tgcgcctacg ccgacgccga cctggtgctg 960 ctcgacaacg ccctgtccgc cgtggaccac cacaccgccc accacatctt cgacaagtgc 1020 atcaagggcc tgttctccga caaggccgtg gtgctggtca cccaccagat cgagttcatg 1080 ccccgctgcg acaacgtggc catcatggac gagggccgct gcctgtactt cggcaagtgg 1140 aacgaggagg cccagcacct gctcggcaag ctgctgccca tcacccacct gctgcacgcc 1200 gccggctccc aggaggctcc ccccgccccc aagaagaagg ccgaggacaa ggccggcccc 1260 cagaagtcgc agtcgctgca gctgaccctg gcccccacct ccatcggcaa gcccaccgag 1320 aagcccaagg acgtccagaa gctgactgcc taccaggccg ccctcatcta cacctggtac 1380 ggcaacctgt tcctggttgg cgtgtgcttc ttcttcttcc tggcggctca gtgctctcgc 1440 cagatctccg atttctgggt gcgctggtgg gtgaacgacg agtacaagaa gttccccgtg 1500 aagggcgagc aggactcggc cgccaccacc ttctactgcc tcatctacct gctgctggtg 1560 ggcctgttct acatcttcat gatcttccgc ggcgccactt tcctgtggtg ggtgctcaag 1620 tcctcggaga ccatccgcag gaaggccctg cacaacgtcc tcaacgcgcc catgggcttc 1680 ttcctggtca cgccggtcgg cgacctgctg ctcaacttca ccaaggacca ggacattatg 1740 gatgagaacc tgcccgatgc cgttcacttc atgggcatct acggcctgat tctgctggcg 1800 accaccatca ccgtgtccgt caccatcaac ttcttcgccg ccttcaccgg cgcgctgatc 1860 atcatgaccc tcatcatgct ctccatctac ctgcccgccg ccactgccct gaagaaggcg 1920 cgcgccgtgt ctggcggcat gctggtcggc ctggttgccg aggttctgga gggccttggc 1980 gtggttcagg ccttcaacaa gcaggagtac ttcattgagg aggccgcccg ccgcaccaac 2040 atcaccaact ccgccgtctt caacgccgag gcgctgaacc tgtggctggc tttctggtgc 2100 gacttcatcg gcgcctgcct ggtgggcgtg gtgtccgcct tcgccgtggg catggccaag 2160 gacctgggcg gcgcgaccgt cggcctggcc ttctccaaca tcattcagat gcttgtgttc 2220 tacacctggg tggtccgctt catctccgag tccatctccc tcttcaactc cgtcgagggc 2280 atggcctacc tcgccgacta cgtgccccac gatggtgtct tctatgacca gcgccagaag 2340 gacggcgtcg ccaagcaaat cgtcctgccc gacggcaaca tcgtgcccgc cgcctccaag 2400 gtccaggtcg tggttgacga cgccgccctc gcccgctggc ctgccaccgg caacatccgc 2460 ttcgaggacg tgtggatgca gtaccgcctg gacgctcctt gggctctgaa gggcgtcacc 2520 ttcaagatca acgacggcga gaaggtcggc gccgtgggcc gcaccggctc cggcaagtcc 2580 accacgctgc tggcgctgta ccgcatgttc gagctgggca agggccgcat cctggtcgac 2640 ggcgtggaca tcgccaccct gtcgctcaag cgcctgcgca ccggcctgtc catcattccc 2700 caggagcccg tcatgttcac cggcaccgtg cgctccaacc tggacccctt cggcgagttc 2760 aaggacgatg ccattctgtg ggaggtgctg aagaaggtcg gcctcgagga ccaggcgcag 2820 cacgccggcg gcctggacgg ccaggtcgat ggcaccggcg gcaaggcctg gtctctgggc 2880 cagatgcagc tggtgtgcct ggctcgcgcc gccctgcgcg ccgtgcccat cctgtgcctg 2940 gacgaggcta ccgccgccat ggacccgcac actgaggcca tcgtgcagca gaccatcaag 3000 aaggtgttcg acgaccgcac caccatcacc attgcccacc gcctggacac catcatcgag 3060 tccgacaaga tcatcgtgat ggagcagggc tcgctgatgg agtacgagtc gccctcgaag 3120 ctgctcgcca accgcgactc catgttctcc aagctggtcg acaagaccgg ccccgccgcc 3180 gccgctgcgc tgcgcaagat ggccgaggac ttctggtcca ctcgctccgc gcagggccgc 3240 aaccagtaa

An exemplary optimized DNA sequence for Chloroplast envelope localized Bicarbonate transporter is shown in SEQ ID NO: 92

(SEQ ID NO: 92) atgcagacca ctatgactcg cccttgcctt gcccagcccg tgctgcgatc tcgtgtgctc 60 cggtcgccta tgcgggtggt tgcagcgagc gctcctaccg cggtgacgac agtcgtgacc 120 tcgaatggaa atggcaacgg tcatttccaa gctgctacta cgcccgtgcc ccctactccc 180 gctcccgtcg ctgtttccgc gcctgtgcgc gctgtgtcgg tgctgactcc tcctcaagtg 240 tatgagaacg ccattaatgt tggcgcctac aaggccgggc taacgcctct ggcaacgttt 300 gtccagggca tccaagccgg tgcctacatt gcgttcggcg ccttcctcgc catctccgtg 360 ggaggcaaca tccccggcgt cgccgccgcc aaccccggcc tggccaagct gctatttgct 420 ctggtgttcc ccgtgggtct gtccatggtg accaactgcg gcgccgagct gttcacgggc 480 aacaccatga tgctcacatg cgcgctcatc gagaagaagg ccacttgggg gcagcttctg 540 aagaactgga gcgtgtccta cttcggcaac ttcgtgggct ccatcgccat ggtcgccgcc 600 gtggtggcca ccggctgcct gaccaccaac accctgcctg tgcagatggc caccctcaag 660 gccaacctgg gcttcaccga ggtgctgtcg cgctccatcc tgtgcaactg gctggtgtgc 720 tgcgccgtgt ggtccgcctc cgccgccacc tcgctgcccg gccgcatcct ggcgctgtgg 780 ccctgcatca ccgccttcgt ggccatcggc ctggagcact ccgtcgccaa catgttcgtg 840 attcctctgg gcatgatgct gggcgctgag gtcacgtgga gccagttctt tttcaacaac 900 ctgatccccg tcaccctggg caacaccatt gctggcgttc tcatgatggc catcgcctac 960 tccatctcgt tcggctccct cggcaagtcc gccaagcccg ccaccgcg 1008

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure specifically described herein. For example a transgenic plant or alga of an embodiment disclosed herein further comprising within its genome, and expressing or overexpressing, a combination of heterologous nucleotide sequences encoding additionally a Rubisco (for example SEQ ID NO:107). Further still a transit peptide amino acid sequence at the amine terminal portion of a protein sequence identified herein may be cleaved leaving the protein sequence alone. The percent homology applies to the protein sequence without the transit peptide sequence also. Such equivalents are intended to be encompassed within the scope of the following claims.

REFERENCES CITED

-   1. Hausler R E, Hirsch H J, Kreuzaler F, Peterhansel C (2002)     Overexpression of C(4)-cycle enzymes in transgenic C(3) plants: a     biotechnological approach to improve C(3)-photosynthesis. J Exp Bot     53: 591-607. -   2. Goldschmidt E E, Huber S C (1992) Regulation of photosynthesis by     end-product accumulation in leaves of plants storing starch,     sucrose, and hexose sugars. Plant Physiol 99: 1443-1448. -   3. Duanmu D, Miller A R, Horken K M, Weeks D P, Spalding M H (2009)     Knockdown of limiting-CO₂-induced gene HLA3 decreases HCO3-transport     and photosynthetic Ci affinity in Chlamydomonas reinhardtii. Proc     Natl Acad Sci USA 106: 5990-5995. -   4. Moroney J V, Jungnick N, Dimario R J, Longstreth D J (2013)     Photorespiration and carbon concentrating mechanisms: two     adaptations to high 02, low CO₂ conditions. Photosynth Res 117:     121-131. -   5. Wang Y, Duanmu D, Spalding M H (2011) Carbon dioxide     concentrating mechanism in Chlamydomonas reinhardtii: inorganic     carbon transport and CO₂ recapture. Photosynth Res 109: 115-122. -   6. Perrine Z, Negi S, Sayre R (2012) Optimization of photosynthetic     light energy utilization by microalgae. Algal Research 1: 134-142. -   7. Elleby B, Chirica L C, Tu C, Zeppezauer M, Lindskog S (2003)     Characterization of carbonic anhydrase from Neisseria gonorrhoeae.     Eur J Biochem 286: 1613-1619. -   8. Subramanian S, Barry A N, Pieris S, Sayre R T (2013) Comparative     energetics and kinetics of autotrophic lipid and starch metabolism     in chlorophytic microalgae: implications for biomass and biofuel     production. Biotechnol Biofuels 6: 150. -   9. Nakamura N, Iwano M, Havaux M, Yokota A, Munekage Y N (2013)     Promotion of cyclic electron transport around photosystem I during     the evolution of NADP-malic enzyme-type C4 photosynthesis in the     genus Flaveria. New Phytol 199: 832-842. -   10. Kramer D M, Evans J R (2011) The importance of energy balance in     improving photosynthetic productivity. Plant Physiol 155: 70-78. -   11. Alric J (2010) Cyclic electron flow around photosystem I in     unicellular green algae. Photosynth Res 106: 47-56. -   12. Amunts A, Drory O, Nelson N (2007) The structure of a plant     photosystem I supercomplex at 3.4 A resolution. Nature 447: 58-63. -   13. Breyton C, Nandha B, Johnson G N, Joliot P, Finazzi G (2006)     Redox modulation of cyclic electron flow around photosystem I in C3     plants. Biochemistry 45: 13465-13475. -   14. Cardol P, Forti G, Finazzi G (2011) Regulation of electron     transport in microalgae. Biochim Biophys Acta 1807: 912-918. -   15. Hanke G T, Okutani S, Satomi Y, Takao T, Suzuki A, et al. (2005)     Multiple iso-proteins of FNR in Arabidopsis: evidence for different     contributions to chloroplast function and nitrogen assimilation.     Plant Cell Environ 28: 1146-1157. -   16. Johnson G N (2011) Physiology of PSI cyclic electron transport     in higher plants. Biochim Biophys Acta 1807: 384-389. -   17. Okutani S, Hanke G T, Satomi Y, Takao T, Kurisu G, et al. (2005)     Three maize leaf ferredoxin:NADPH oxidoreductases vary in     subchloroplast location, expression, and interaction with     ferredoxin. Plant Physiol 139: 1451-1459. -   18. Slewinski T L, Braun D M (2010) Current perspectives on the     regulation of whole-plant carbohydrate partitioning. Plant Science     178: 341-349. -   19. Arrivault S, Guenther M, Ivakov A, Feil R, Vosloh D, et     al. (2009) Use of reverse-phase liquid chromatography, linked to     tandem mass spectrometry, to profile the Calvin cycle and other     metabolic intermediates in Arabidopsis rosettes at different carbon     dioxide concentrations. Plant Journal 59: 824-839. -   20. Huege J, Sulpice R, Gibon Y, Lisec J, Koehl K, et al. (2007)     GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble     metabolite pools of higher plants by monitoring isotope dilution     after (CO₂)—C-13 labelling. Phytochemistry 68: 2258-2272. -   21. Romisch-Margl W, Schramek N, Radykewicz T, Ettenhuber C, Eylert     E, et al. (2007) (CO₂)—C-13 as a universal metabolic tracer in     isotopologue perturbation experiments. Phytochemistry 68: 2273-2289. -   22. Sekiyama Y, Kikuchi J (2007) Towards dynamic metabolic network     measurements by multi-dimensional NMR-based fluxomics.     Phytochemistry 68: 2320-2329. -   23. Szecowka M, Heise R, Tohge T, Nunes-Nesi A, Vosloh D, et     al. (2013) Metabolic fluxes in an illuminated Arabidopsis rosette.     Plant Cell 25: 694-714. -   24. Ma F, Jazmin L J, Young J D, Allen D K (Submitted) Isotopically     nonstationary 13C flux analysis of Arabidopsis thaliana leaf     metabolism at varying light intensities. Proc Natl Acad Sci USA. -   25. Shastri A A, Morgan J A (2007) A transient isotopic labeling     methodology for 13C metabolic flux analysis of photoautotrophic     microorganisms. Phytochemistry 68: 2302-2312. -   26. Young J D, Shastri A A, Stephanopoulos G, Morgan J A (2011)     Mapping photoautotrophic metabolism with isotopically nonstationary     (13)C flux analysis. Metab Eng 13: 656-665. -   27. Young J D (Submitted) INCA: A computational platform for     isotopically nonstationary metabolic flux analysis. Bioinformatics. -   28. Young J D, Walther J L, Antoniewicz M R, Yoo H, Stephanopoulos     G (2008) An elementary metabolite unit (EMU) based method of     isotopically nonstationary flux analysis. Biotechnol Bioeng 99:     686-699. -   29. Masclaux-Daubresse C, Chardon F (2011) Exploring nitrogen     remobilization for seed filling using natural variation in     Arabidopsis thaliana. J Exp Bot 62: 2131-2142. -   30. Hay R K M, Gilbert R A (2001) Variation in the harvest index of     tropical maize: Evaluation of recent evidence from Mexico and     Malawi. Annals of Applied Biology 138: 103-109. -   31. Russell W A (1985) Evaluation for plant, ear and grain traits of     maize cultivars representing seven years of breeding. Maydica 30:     85-96. -   32. Sinclair T R (1998) Historical changes in harvest index and crop     nitrogen accumulation. Crop Science 38: 638-643. -   33. Victorio R G, Moreno U, Black Jr C C (1986) Growth,     partitioning, and harvest index of tuber-bearing Solanum genotypes     grown in two contrasting Peruvian environments. Plant Physiology 82:     103-108. -   34. Vos J (1997) The nitrogen response of potato (Solanum tuberosum     L.) in the field: Nitrogen uptake and yield, harvest index and     nitrogen concentration. Potato Research 40: 237-248. -   35. Parry M A, Andralojc P J, Scales J C, Salvucci M E, Carmo-Silva     A E, et al. (2013) Rubisco activity and regulation as targets for     crop improvement. J Exp Bot 64: 717-730. -   36. Sage R F (2002) Variation in the k(cat) of Rubisco in C(3) and     C(4) plants and some implications for photosynthetic performance at     high and low temperature. J Exp Bot 53: 609-620. -   37. Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M (2001) A     small decrease of plastid transketolase activity in antisense     tobacco transformants has dramatic effects on photosynthesis and     phenylpropanoid metabolism. Plant Cell 13: 535-551. -   38. Miyagawa Y, Tamoi M, Shigeoka S (2001) Overexpression of a     cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in     tobacco enhances photosynthesis and growth. Nature Biotechnology 19:     965-969. -   39. Peterhansel C, Blume C, Offermann S (2013) Photorespiratory     bypasses: how can they work? J Exp Bot 64: 709-715. -   40. Blanco N E, Ceccoli R D, Via M V, Voss I, Segretin M E, et     al. (2013) Expression of the minor isoform pea ferredoxin in tobacco     alters photosynthetic electron partitioning and enhances cyclic     electron flow. Plant Physiol 161: 866-879. -   41. Busch K B, Deckers-Hebestreit G, Hanke G T, Mulkidjanian A     Y (2012) Dynamics of bioenergetic microcompartments. Biol Chem 394:     163-188. -   42. Minagawa J (2011) State transitions—the molecular remodeling of     photosynthetic supercomplexes that controls energy flow in the     chloroplast. Biochim Biophys Acta 1807: 897-905. -   43. Peltier G, Tolleter D, Billon E, Cournac L (2010) Auxiliary     electron transport pathways in chloroplasts of microalgae.     Photosynth Res 106: 19-31. -   44. Peng L, Shikanai T (2011) Supercomplex formation with     photosystem I is required for the stabilization of the chloroplast     NADH dehydrogenase-like complex in Arabidopsis. Plant Physiol 155:     1629-1639. -   45. Takahashi H, Clowez S, Wollman F A, Vallon O, Rappaport F (2013)     Cyclic electron flow is redox-controlled but independent of state     transition. Nat Commun 4: 1954. -   46. Neale A P, Blunder T, Wunder T, Pesaresi P, Pribil M, et     al. (2013) PGRL1 is the elusive ferredoxin-plastoquinone reductase     in photosynthetic cyclic electron flow. Mol Cell 49: 511-523. -   47. DalCorso G, Pesaresi P, Masiero S, Aseeva E, Schunemann D, et     al. (2008) A complex containing PGRL1 and PGR5 is involved in the     switch between linear and cyclic electron flow in Arabidopsis. Cell     132: 273-285. -   48. Shikanai T (2014) Central role of cyclic electron transport     around photosystem I in the regulation of photosynthesis. Current     Opinion in Biotechnology 26: 25-30. -   49. Walter J M, Greenfield D, Liphardt J (2010) Potential of     light-harvesting proton pumps for bioenergy applications. Curr Opin     Biotechnol 21: 265-270. -   50. Dioumaev A K, Brown L S, Shih J, Spudich E N, Spudich J L, et     al. (2002) Proton transfers in the photochemical reaction cycle of     proteorhodopsin. Biochemistry 41: 5348-5358. -   51. Friedrich T, Geibel S, Kalmbach R, Chizhov I, Ataka K, et     al. (2002) Proteorhodopsin is a light-driven proton pump with     variable vectoriality. J Mol Biol 321: 821-838. -   52. Govindjee R, Ebrey T G, Crofts A R (1980) The quantum efficiency     of proton pumping by the purple membrane of Halobacterium halobium.     Biophys J 30: 231-242. -   53. Govindjee R, Imasheva E S, Misra S, Balashov S P, Ebrey T G, et     al. (1997) Mutation of a surface residue, lysine-129, reverses the     order of proton release and uptake in bacteriorhodopsin; guanidine     hydrochloride restores it. Biophys J 72: 886-898. -   54. Govindjee R, Misra S, Balashov S P, Ebrey T G, Crouch R K, et     al. (1996) Arginine-82 regulates the pKa of the group responsible     for the light-driven proton release in bacteriorhodopsin. Biophys J     71: 1011-1023. -   55. Lakatos M, Lanyi J K, Szakacs J, Varo G (2003) The photochemical     reaction cycle of proteorhodopsin at low pH. Biophys J 84:     3252-3256. -   56. Walter J M, Greenfield D, Bustamante C, Liphardt J (2007)     Light-powering Escherichia coli with proteorhodopsin. Proc Natl Acad     Sci USA 104: 2408-2412. -   57. Kim J Y, Jo B H, Jo Y, Cha H J (2012) Improved production of     biohydrogen in light-powered Escherichia coli by co-expression of     proteorhodopsin and heterologous hydrogenase. Microb Cell Fact 11:2. -   58. Froehlich J E, Keegstra K (2011) The role of the transmembrane     domain in determining the targeting of membrane proteins to either     the inner envelope or thylakoid membrane. Plant J 68: 844-856. -   59. Beja O, Aravind L, Koonin E V, Suzuki M T, Hadd A, et al. (2000)     Bacterial rhodopsin: evidence for a new type of phototrophy in the     sea. Science 289: 1902-1906. -   60. Lindqvist A, Andersson S (2002) Biochemical properties of     purified recombinant human beta-carotene 15,15′-monooxygenase. J     Biol Chem 277: 23942-23948. -   61. Roslan H A, Salter M G, Wood C D, White M R, Croft K P, et     al. (2001) Characterization of the ethanol-inducible alc     gene-expression system in Arabidopsis thaliana. Plant J 28: 225-235. -   62. Cao Y, Brown L S, Sasaki J, Maeda A, Needleman R, et al. (1995)     Relationship of proton release at the extracellular surface to     deprotonation of the schiff base in the bacteriorhodopsin     photocycle. Biophys J 68: 1518-1530. -   63. Joliot P, Johnson G N (2011) Regulation of cyclic and linear     electron flow in higher plants. Proc Natl Acad Sci USA 108:     13317-13322. -   64. Fabre N, Reiter I M, Becuwe-Linka N, Genty B, Rumeau D (2007)     Characterization and expression analysis of genes encoding alpha and     beta carbonic anhydrases in Arabidopsis. Plant Cell Environ 30:     617-629. -   65. Bihmidine S, Hunter C T, 3rd, Johns C E, Koch K E, Braun D     M (2013) Regulation of assimilate import into sink organs: update on     molecular drivers of sink strength. Front Plant Sci 4: 177. -   66. Ihemere U, Arias-Garzon D, Lawrence S, Sayre R (2006) Genetic     modification of cassava for enhanced starch production. Plant     Biotechnol J 4: 453-465. -   67. Wunsche J N, Greer D H, Laing W A, Palmer J W (2005)     Physiological and biochemical leaf and tree responses to crop load     in apple. Tree Physiol 25: 1253-1263. -   68. Paul M J, Foyer C H (2001) Sink regulation of photosynthesis. J     Exp Bot 52: 1383-1400. -   69. Sonnewald U, Lerchi J, Zrenner R, Frommer W (1994) Manipulation     of sink-source relations in transgenic plants. Plant Cell Environ     17: 649-658. -   70. Sonnewald U, Willmitzer L (1992) Molecular approaches to     sink-source interactions. Plant Physiol 99: 1267-1270. -   71. Willson W J (1972) Control of crop processes In: Rees A R,     Cockshull K E, Hand D W, Hurd R G, editors. Crop Processes in     Controlled Environments: London Academic Press. pp. 7-30. -   72. Jonik C, Sonnewald U, Hajirezaei M R, Flugge U I, Ludewig     F (2012) Simultaneous boosting of source and sink capacities doubles     tuber starch yield of potato plants. Plant Biotechnol J 10:     1088-1098. -   73. Sweetlove L J, Hill S A (2000) Source metabolism dominates the     control of source to sink carbon flux in tuberizing potato plants     throughout the diurnal cycle and under a range of environmental     conditions. Plant, Cell and Environment 23: 523-529. -   74. Allen D K, Goldford J, Gierse J, Mandy D, Diepenbrock C, et     al. (2013) (submitted) Quantification of peptide m/z distributions     form 13C-labeled cultures with high resolution mass spectrometry.     Analytical Chemistry. -   75. Choi J, Antoniewicz M R (2011) Tandem mass spectrometry: a novel     approach for metabolic flux analysis. Metab Eng 13: 225-233. -   76. Allen D K, Libourel I G L, Shachar-Hill Y (2009) Metabolic flux     analysis in plants: Coping with complexity. Plant, Cell and     Environment 32: 1241-1257. -   77. Allen D K, Laclair R W, Ohlrogge J B, Shachar-Hill Y (2012)     Isotope labelling of Rubisco subunits provides in vivo information     on subcellular biosynthesis and exchange of amino acids between     compartments. Plant, Cell and Environment 35: 1232-1244. -   78. Allen D K, Shachar-Hill Y, Ohlrogge J B (2007)     Compartment-specific labeling information in ¹³C metabolic flux     analysis of plants. Phytochemistry 68: 2197-2210. -   79. Mandy D, Goldford J, Yang H, Allen D K, Libourel I G L (2013)     (submitted) Metabolic flux analysis using 13C peptide label     measurements. The Plant Journal. -   80. Allen D K, Young J D (2013) Carbon and nitrogen provisions alter     the metabolic flux in developing soybean embryos. Plant Physiol 161:     1458-1475. -   81. Allen D K, Ohlrogge J B, Shachar-Hill Y (2009) The role of light     in soybean seed filling metabolism. Plant Journal 58: 220-234. -   82. Jazmin L J, Young J D (2013) Isotopically nonstationary 13C     metabolic flux analysis. Methods Mol Biol 985: 367-390. -   83. Blankenship R E, Tiede D M, Barber J, Brudvig G W, Fleming G, et     al. (2011) Comparing photosynthetic and photovoltaic efficiencies     and recognizing the potential for improvement. Science 332: 805-809. -   84. Jazmin L J, O'Grady J, Ma F, Allen D K, Morgan J A, et al. (In     press) Isotopically nonstationary MFA (INST-MFA) of autotrophic     metabolism. Methods Mol Biol. -   85. Egnatchik R A, Leamy A K, Noguchi Y, Shiota M, Young J D (In     press) Palmitate-induced activation of mitochondrial metabolism     promotes oxidative stress and apoptosis in H411EC3 rat hepatocytes.     Metabolism. -   86. Leamy A K, Egnatchik R A, Young J D (2013) Molecular mechanisms     and the role of saturated fatty acids in the progression of     non-alcoholic fatty liver disease. Prog Lipid Res 52: 165-174. -   87. Srour O, Young J D, Eldar Y C (2011) Fluxomers: a new approach     for 13C metabolic flux analysis. BMC Syst Biol 5: 129. -   88. Young J D, Allen D K, Morgan J A (2014) Isotopomer measurement     techniques in metabolic flux analysis II: Mass spectrometry. Methods     Mol Biol 1083: 85-108. -   89. Egnatchik R A, Leamy A K, Jacobson D A, Young J D (Submitted) E     R calcium stimulates mitochondrial alterations in hepatic     lipotoxicity. J Biol Chem. -   90. Leamy A K, Egnatchik R A, Shiota M, Young J D (Submitted)     Modulating lipid fate controls E R stress and lipotoxicity in     palmitate-treated hepatic cells. FEBS J. -   91. Young J D (In press) Metabolic flux rewiring in mammalian cell     cultures. Curr Opin Biotechnol. -   92. McAtee A G, Templeton N, Young J D (Submitted) Role of CHO     central carbon metabolism in controlling the quality of secreted     biotherapeutic proteins. Pharmaceutical Bioprocessing. -   93. Duckwall C S, Murphy T A, Young J D (2013) Mapping cancer cell     metabolism with (13)C flux analysis: Recent progress and future     challenges. J Carcinog 12: 13. 

What is claimed is:
 1. A construct comprising: i) a first heterologous nucleic acid sequence comprising a first heterologous polynucleotide sequence encoding a cyclic electron modulator gene wherein the cyclic electron modulator is operatively linked to at least one regulatory element wherein the first heterologous nucleic acid sequence encodes a protein having a sequence selected from PRG5 of SEQ ID NO: 1 or PGRL1 of SEQ ID NO: 3 or a sequence with at least 80% sequence homology thereto; and ii) a second heterologous nucleic acid sequence comprising a second heterologous polynucleotide sequence encoding an ATP dependent bicarbonate anion transporter localized to the plasma membrane wherein the ATP dependent bicarbonate anion transporter localized to the plasma membrane is operatively coupled to the at least one regulatory element wherein the second heterologous nucleic acid sequence encodes a protein having a sequence selected from HLA3 (SEQ ID NO:77) or a sequence with at least 80% sequence homology thereto.
 2. The construct of claim 1 wherein the HLA3 is codon optimized for plant expression.
 3. The construct of claim 1 wherein the at least one regulatory element includes a promoter.
 4. The construct of claim 1 wherein the at least one regulatory element is a tissue specific promoter.
 5. The construct of claim 1 wherein the at least one regulatory element includes a promoter that is a green tissue/leaf-specific promoter.
 6. The construct of claim 1 wherein the promoter is selected from among CAB and rbcS.
 7. The construct of claim 1 wherein the first nucleic acid sequence and the second nucleic acid sequence encode: i) the PGR5 protein, and the HLA3 protein; or ii) the PGRL1 protein, and the HLA3 protein.
 8. The construct of claim 1 further comprising iii) a third heterologous nucleic acid sequence comprising a third heterologous polynucleotide sequence encoding a bicarbonate anion transporter protein localized to the chloroplast envelope wherein the bicarbonate anion transporter protein localized to the chloroplast envelope is operatively coupled to the regulatory element wherein the third heterologous nucleic acid sequence i-encodes a sequence selected from LCIA of SEQ ID NO: 18 or a sequence with at least 80% sequence homology thereto.
 9. The construct of claim 8 wherein the at least one regulatory element is a green tissue/leaf-specific promoter.
 10. The construct of claim 8 wherein the at least one regulatory element includes a promoter selected from among CAB and rbcS.
 11. The construct of claim 1 further comprising iii) a third heterologous nucleic acid sequence comprising a third heterologous polynucleotide sequence encoding a carbonic anhydrase wherein the carbonic anhydrase is operatively coupled to the at least one regulatory element wherein the third heterologous nucleic acid sequence encodes a protein selected from a human carbonic anhydrase-2 (HCA2) of SEQ ID NO: 18, bacterial Neisseria gonorrhoeae carbonic anhydrase (BCA) of SEQ ID NO: 5 or a sequence with at least 80% sequence homology thereto.
 12. The construct of claim 11 wherein the at least one regulatory element includes a green tissue/leaf-specific promoter.
 13. The construct of claim 11 wherein the at least one regulatory element includes a promoter selected from among CAB and rbcS.
 14. The construct of claim 11 wherein the third heterologous nucleic acid sequence encodes the BCA protein of SEQ ID NO: 5 protein or a sequence with at least 80% sequence homology thereto.
 15. The construct of claim 8 wherein the heterologous nucleotide sequences encode the PGR5 protein, the HLA3 protein, and the LCIA protein or sequences with at least 80% homology thereto.
 16. The construct of claim 11 wherein the heterologous nucleotide sequences encode the PGR5 protein, the HLA3 protein, and the BCA protein or sequences with at least 80% homology thereto.
 17. The construct of claim 11, wherein a) the PGR5 protein has an amino acid sequence at least 80% identical to SEQ ID NO:1; b) the HLA 3 protein has an amino acid sequence at least 80% identical to SEQ ID NO:77; and c) the BCA protein has an amino acid sequence at least 80% identical to SEQ ID NO:21.
 18. The construct of claim 1 wherein the at least one regulatory element of the first heterologous nucleic acid sequence and the at least one regulatory element of the second heterologous nucleic acid sequence includes a promoter which can be the same or different for the first heterologous nucleic acid and the second heterologous nucleic acid.
 19. A seed comprising the construct of claim
 1. 20. A vector comprising the construct of claim
 1. 